Metalloproteinase inhibitor compounds

ABSTRACT

Compounds of the formula I 
                         
useful as metalloproteinase inhibitors, especially as inhibitors of MMP 13.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.09/788,687, filed Feb. 21, 2001, now U.S. Pat. No. 6,734,183 whichclaims priority from United Kingdom Application No. 00400467.7, filedFeb. 21, 2000, the specifications of each of which are incorporated byreference herein.

The present invention relates to compounds useful in the inhibition ofmetalloproteinases and in particular to pharmaceutical compositionscomprising these, as well as their use.

The compounds of this invention are inhibitors of one or moremetalloproteinase enzymes. Metalloproteinases are a superfamily ofproteinases (enzymes) whose numbers in recent years have increaseddramatically. Based on structural and functional considerations theseenzymes have been classified into families and subfamilies as describedin N. M. Hooper (1994) FEBS Letters 354:1–6. Examples ofmetalloproteinases include the matrix metalloproteinases (MMP) such asthe coliagenases (MMP1, MMP8, MMP13), the gelatinases (MMP2, MMP9), thestromelysins (MMP3, MMP10, MMP11), matrilysin (MMP7), metalloelastase(MMP12), enamelysin (MP19), the MT-MMPs (MMP14, MMP15, MMP16, MMP17);the reprolysin or adamalysin or MDC family which includes the secretasesand sheddases such as TNF converting enzymes (ADAM10 and TACE); theastacin family which include enzymes such as procollagen processingproteinase (PCP); and other metalloproteinases such as aggrecanase, theendothelin converting enzyme family and the angiotensin convertingenzyme family.

Metalloproteinases are believed to be important in a plethora ofphysiological disease processes that involve tissue remodelling such asembryonic development, bone formation and uterine remodelling duringmenstruation. This is based on the ability of the metalloproteinases tocleave a broad range of matrix substrates such as collagen, proteoglycanand fibronectin. Metalloproteinases are also believed to be important inthe processing, or secretion, of biological important cell mediators,such as tumour necrosis factor (TNF); and the post translationalproteolysis processing, or shedding, of biologically important membraneproteins, such as the low affinity IgE receptor CD23 (for a morecomplete list see N. M. Hooper et al., (1997) Biochem J. 321:265–279).

Metalloproteinases have been associated with many disease conditions.Inhibition of the activity of one or more metalloproteinases may well beof benefit in these disease conditions, for example: variousinflammatory and allergic diseases such as, inflammation of the joint(especially rheumatoid arthritis, osteoarthritis and gout), inflammationof the gastro-intestinal tract (especially inflammatory bowel disease,ulcerative colitis and gastritis), inflammation of the skin (especiallypsoriasis, eczema, dermatitis); in tumour metastasis or invasion; indisease associated with uncontrolled degradation of the extracellularmatrix such as osteoarthritis; in bone resorptive disease (such asosteoporosis and Paget's disease); in diseases associated with aberrantangiogenesis; the enhanced collagen remodelling associated withdiabetes, periodontal disease (such as gingivitis), corneal ulceration,ulceration of the skin, post-operative conditions (such as colonicanastomosis) and dermal wound healing; demyelinating diseases of thecentral and peripheral nervous systems (such as multiple sclerosis);Alzheimer's disease; extracellular matrix remodelling observed incardiovascular diseases such as restenosis and atheroscelerosis; andchronic obstructive pulmonary diseases, COPD (for example, the role ofMMPs such as MMP12 is discussed in Anderson & Shinagawa, 1999, CurrentOpinion in Anti-inflammatory and Immunomodulatory Investigational Drugs,1(1): 29–38).

A number of metalloproteinase inhibitors are known; different classes ofcompounds may have different degrees of potency and selectivity forinhibiting various metalloproteinases. We have discovered a new class ofcompounds that are inhibitors of metalloproteinases and are ofparticular interest in inhibiting MMP-13, as well as MMP-9. Thecompounds of this invention have beneficial potency and/orpharmacokinetic properties.

MMP13, or collagenase 3, was initially cloned from a cDNA libraryderived from a breast tumour [J. M. P. Freije et al. (1994) Journal ofBiological Chemistry 269(24):16766–16773]. PCR-RNA analysis of RNAs froma wide range of tissues indicated that MMP13 expression was limited tobreast carcinomas as it was not found in breast fibroadenomas, normal orresting mammary gland, placenta, liver, ovary, uterus, prostate orparotid gland or in breast cancer cell lines (T47-D, MCF-7 and ZR75-1).Subsequent to this observation MMP13 has been detected in transformedepidermal keratinocytes [N. Johansson et al., (1997) Cell Growth Differ.8(2):243–250], squamous cell carcinomas [N. Johansson et al., (1997) Am.J. Pathol. 151(2):499–508] and epidermal tumours [K. Airola et al.,(1997) J. Invest. Dermatol. 109(2):225–231]. These results aresuggestive that MMP13 is secreted by transformed epithelial cells andmay be involved in the extracellular matrix degradation and cell-matrixinteraction associated with metastasis especially as observed ininvasive breast cancer lesions and in malignant epithelia growth in skincarcinogenesis.

Recent published data implies that MMP13 plays a role in the turnover ofother connective tissues. For instance, consistent with MMP13'ssubstrate specificity and preference for degrading type II collagen [P.G. Mitchell et al., (1996) J. Clin. Invest. 97(3):761–768; V. Knauper etal., (1996) The Biochemical Journal 271:1544–1550], MMP13 has beenhypothesised to serve a role during primary ossification and skeletalremodelling [M. Stahle-Backdahl et al., (1997) Lab. Invest.76(5):717–728; N. Johansson et al., (1997) Dev. Dyn. 208(3):387–397], indestructive joint diseases such as rheumatoid and osteo-arthritis [D.Wernicke et al., (1996) J. Rheumatol. 23:590–595; P. G. Mitchell et al.,(1996) J. Clin. Invest. 97(3):761–768; O. Lindy et al., (1997) ArthritisRheum 40(8):1391–1399]; and during the aseptic loosening of hipreplacements [S. Imai et al., (1998) J. Bone Joint Surg. Br.80(4):701–710]. MMP13 has also been implicated in chronic adultperiodontitis as it has been localised to the epithelium of chronicallyinflamed mucosa human gingival tissue [V. J. Uitto et al., (1998) Am. J.Pathol 152(6):1489–1499] and in remodelling of the collagenous matrix inchronic wounds [M. Vaalamo et al., (1997) J. Invest. Dermatol.109(1):96–101].

MMP9 (Gelatinase B; 92 kDa TypeIV Collagenase; 92 kDa Gelatinase) is asecreted protein which was first purified, then cloned and sequenced, in1989 (S. M. Wilhelm et al (1989) J. Biol Chem. 264 (29): 17213–17221.Published erratum in J. Biol Chem. (1990) 265 (36): 22570). A recentreview of MMP9 provides an excellent source for detailed information andreferences on this protease: T. H. Vu & Z. Werb (1998) (In: MatrixMetalloproteinases. 1998. Edited by W. C. Parks & R. P. Mecham.pp115–148. Academic Press. ISBN 0-12-545090-7). The following points aredrawn from that review by T. H. Vu & Z. Werb (1998).

The expression of MMP9 is restricted normally to a few cell types,including trophoblasts, osteoclasts, neutrophils and macrophages.However, it's expression can be induced in these same cells and in othercell types by several mediators, including exposure of the cells togrowth factors or cytokines. These are the same mediators oftenimplicated in initiating an inflammatory response. As with othersecreted MMPs, MMP9 is released as an inactive Pro-enzyme which issubsequently cleaved to form the enzymatically active enzyme. Theproteases required for this activation in vivo are not known. Thebalance of active MMP9 versus inactive enzyme is further regulated invivo by interaction with TIMP-1 (Tissue Inhibitor ofMetalloproteinases-1), a naturally-occurring protein. TIMP-1 binds tothe C-terminal region of MMP9, leading to inhibition of the catalyticdomain of MMP9. The balance of induced expression of ProMMP9, cleavageof Pro- to active MMP9 and the presence of TIMP-1 combine to determinethe amount of catalytically active MMP9 which is present at a localsite. Proteolytically active MMP9 attacks substrates which includegelatin, elastin, and native Type IV and Type V collagens; it has noactivity against native Type I collagen, proteoglycans or lamanins.

There has been a growing body of data implicating roles for MMP9 invarious physiological and pathological processes. Physiological rolesinclude the invasion of embryonic trophoblasts through the uterineepithelium in the early stages of embryonic implantation; some role inthe growth and development of bones; and migration of inflammatory cellsfrom the vasculature into tissues. Increased MMP9 expression hasobserved in certain pathological conditions, thereby implicating MMP9 indisease processed such as arthritis, tumour metastasis, Alzheimer's,Multiple Sclerosis, and plaque rupture in atherosclerosis leading toacute coronary conditions such as Myocardial Infarction.

WO-99/38843 claims compounds of the general formulaB—X—(CH₂)_(m)—(CR¹R²)_(n)—W—COYfor use in the manufacture of a medicament for the treatment orprevention of a condition associated with matrix metalloproteinases.Specifically disclosed is the compoundN-{1S-[4-(4-Chlorophenyl)piperazine-1-sulfonylmethyl]-2-methylpropyl}-N-hydroxyformamide.

We have now discovered compounds that are potent MMP13 inhibitors andhave desirable activity profiles.

In a first aspect of the invention we now provide compounds of theformula I

wherein B represents a phenyl group monosubstituted at the 3- or4-position by halogen or trifluoromethyl, or disubstituted at the 3- and4-positions by halogen (which may be the same or different); or Brepresents a 2-pyridyl or 2-pyridyloxy group monosubstituted at the 4-,5- or 6-position by halogen, trifluoromethyl, cyano or C1–4 alkyl; or Brepresents a 4-pyrimidinyl group optionally substituted at the6-position by halogen or C1–4 alkyl;

-   X represents a carbon or nitrogen atom;-   R1 represents a trimethyl-1-hydantoin C2–4alkyl or a    trimethyl-3-hydantoin C2–4alkyl group; phenyl or C2–4alkylphenyl    monosubstituted at the 3- or 4-position by halogen, trifluoromethyl,    thio or C1–3alkyl or C1–3alkoxy; phenyl-SO2NHC2–4alkyl; 2-pyridyl or    2-pyridyl C2–4alkyl; 3-pyridyl or 3-pyridyl C2–4alkyl;    2-pyrimidine-SCH2CH2; 2- or 4-pyrimidinyl C2–4alkyl optionally    monosubstituted by one of halogen, trifluoromethyl, C1–3alkyl,    C1–3alkyloxy, 2-pyrazinyl optionally substituted by halogen or    2-pyrazinyl C2–4alkyl optionally substituted by halogen;    Any alkyl groups outlined above may be straight chain or branched.

Preferred compounds of the invention are those wherein any one or moreof the following apply:

B represents 4-chlorophenyl, 4-fluorophenyl, 4-bromophenyl or4-trifluorophenyl; 2-pyridyl or 2-pyridyloxy monosubstituted at the 4-or 5-position such as 5-chloro-2-pyridyl, 5-bromo-2-pyridyl,5-fluoro-2-pyridyl, 5-trifluoromethyl-2-pyridyl, 5-cyano-2-pyridyl,5-methyl-2-pyridyl; especially 4-fluorophenyl, 5-chloro-2-pyridyl or5-trifluoromethyl-2-pyridyl;

X represents a nitrogen atom;

R1 is 3-chlorophenyl, 4-chlorophenyl, 3-pyridyl, 2-pyridylpropyl, 2- or4-pyrimidinylethyl (optionally monosubstituted by fluorine), 2- or4-pyrimidinylpropyl, 2-(2-pyrimidinyl)propyl (optionally monosubstituedby fluorine); especially 2-pyrimidinylpropyl, 2-(2-pyrimidinyl)propyl(optionally monosubstitued by fluorine) or 5-fluoro-2-pyrimidinylethyl.

For compounds of formula I, a particular subgroup is represented bycompounds wherein B is a phenyl group monosubstituted at the 3- or4-position by halogen or trifluoromethyl, or disubstituted at the 3- and4-positions by halogen (which may be the same or different); or B is a2-pyridyl or 2-pyridyloxy group monosubstituted at the 5- or 6-positionby halogen, trifluoromethyl or cyano; or B is a 4-pyrimidinyl groupoptionally substituted at the 6-position by halogen or C1–4alkyl; X is acarbon or nitrogen atom; R1 is a trimethyl-1-hydantoin C2–4alkyl or atrimethyl-3-hydantoin C2–4alkyl group; or R1 is a phenyl orC2–4alkylphenyl monosubstituted at the 3- or 4-position by halogen,trifluoromethyl, thio or C1–3alkyl or C1–3alkoxy; or R1 isphenyl-SO2NHC2–4alkyl; or R1 is 2-pyridyl or 2-pyridyl C2–4alkyl; or R1is 3-pyridyl or 3-pyridyl C2–4alkyl; or R1 is 2-pyrimidine-SCH2CH2; orR1 is 2- or 4-pyrimidinyl C2–4alkyl optionally monosubstituted by one ofhalogen, trifluoromethyl, C1–3alkyl, C 1–3 alkyloxy, 2-pyrazinyl or2-pyrazinyl C2–4alky; any alkyl group may be straight chain or branched.

It will be appreciated that the particular substituents and number ofsubstituents on B and/or R1 are selected so as to avoid stericallyundesirable combinations.

Each exemplified compound represents a particular and independent aspectof the invention.

Where optically active centres exist in the compounds of formula I, wedisclose all individual optically active forms and combinations of theseas individual specific embodiments of the invention, as well as theircorresponding racemates. Racemates may be separated into individualoptically active forms using known procedures (cf. Advanced OrganicChemistry: 3rd Edition: author J March, p104–107) including for examplethe formation of diastereomeric derivatives having convenient opticallyactive auxiliary species followed by separation and then cleavage of theauxiliary species.

It will be appreciated that the compounds according to the invention cancontain one or more asymmetrically substituted carbon atoms. Thepresence of one or more of these asymmetric centres (chiral centres) ina compound of formula I can give rise to stereoisomers, and in each casethe invention is to be understood to extend to all such stereoisomers,including enantiomers and diastereomers, and mixtures including racemicmixtures thereof.

In the examples we disclose the isolation and characterisation ofcertain enantiomers. Enantiomers may be prepared by the reaction ofracemic material with a chiral auxilliary, separation of thediastereomers formed using chromatography, followed by subsequentcleavage of the chiral auxilliary. The diastereomer eluted second fromthe column (using conditions herein described) and subsequently cleavedgives the more active enantiomer when tested. In each case we believethe active enantiomer has S stereochemistry but do not wish to belimited by this initial determination. The active enantiomer ischaracterised by its derivative being eluted second from the separationcolumn. Use of different compounds of formula I, alternative columnsand/or different solvents may affect the elution order of the mostactive enantiomer.

In the examples we disclose the isolation and characterisation ofcertain diastereomers. Chromatographic separation and subsequent testingrevealed that the more active diastereomer is eluted first from theseparation column (ie the more active diastereomer is characterised bybeing eluted first from the separation column). Use of differentcompounds of formula I, alternative columns and/or different solventsmay affect the elution order of the most active diastereomer.

For compounds of formula I with two chiral centres we believe the activeenantiomer has S,S stereochemistry but do not wish to be limited by thisinitial determination.

Where tautomers exist in the compounds of formula I, we disclose allindividual tautomeric forms and combinations of these as individualspecific embodiments of the invention.

As previously outlined the compounds of the invention aremetalloproteinase inhibitors, in particular they are inhibitors ofMMP13. Each of the above indications for the compounds of the formula Irepresents an independent and particular embodiment of the invention.Whilst we do not wish to be bound by theoretical considerations, thecompounds of the invention are believed to show selective inhibition forany one of the above indications relative to any MMP1 inhibitoryactivity, by way of non-limiting example they may show 100–1000 foldselectivity over any MMP1 inhibitory activity.

Certain compounds of the invention are of particular use as aggrecanaseinhibitors ie. inhibitors of aggrecan degradation. Certain compounds ofthe invention are of particular use as inhibitors of MMP9 and/or MMP12.

The compounds of the invention may be provided as pharmaceuticallyacceptable salts. These include acid addition salts such ashydrochloride, hydrobromide, citrate and maleate salts and salts formedwith phosphoric and sulphuric acid. In another aspect suitable salts arebase salts such as an alkali metal salt for example sodium or potassium,an alkaline earth metal salt for example calcium or magnesium, ororganic amine salt for example triethylamine.

They may also be provided as in vivo hydrolysable esters. These arepharmaceutically acceptable esters that hydrolyse in the human body toproduce the parent compound. Such esters can be identified byadministering, for example intravenously to a test animal, the compoundunder test and subsequently examining the test animal's body fluids.Suitable in vivo hydrolysable esters for carboxy include methoxymethyland for hydroxy include formyl and acetyl, especially acetyl.

In order to use a compound of the formula I or a pharmaceuticallyacceptable salt or in vivo hydrolysable ester thereof for thetherapeutic treatment (including prophylactic treatment) of mammalsincluding humans, it is normally formulated in accordance with standardpharmaceutical practice as a pharmaceutical composition.

Therefore in another aspect the present invention provides apharmaceutical composition which comprises a compound of the formula Ior a pharmaceutically acceptable salt or an in vivo hydrolysable esterand pharmaceutically acceptable carrier.

The pharmaceutical compositions of this invention may be administered instandard manner for the disease condition that it is desired to treat,for example by oral, topical, parenteral, buccal, nasal, vaginal orrectal administration or by inhalation. For these purposes the compoundsof this invention may be formulated by means known in the art into theform of, for example, tablets, capsules, aqueous or oily solutions,suspensions, emulsions, creams, ointments, gels, nasal sprays,suppositories, finely divided powders or aerosols for inhalation, andfor parenteral use (including intravenous, intramuscular or infusion)sterile aqueous or oily solutions or suspensions or sterile emulsions.

In addition to the compounds of the present invention the pharmaceuticalcomposition of this invention may also contain, or be co-administered(simultaneously or sequentially) with, one or more pharmacologicalagents of value in treating one or more disease conditions referred tohereinabove.

The pharmaceutical compositions of this invention will normally beadministered to humans so that, for example, a daily dose of 0.5 to 75mg/kg body weight (and preferably of 0.5 to 30 mg/kg body weight) isreceived. This daily dose may be given in divided doses as necessary,the precise amount of the compound received and the route ofadministration depending on the weight, age and sex of the patient beingtreated and on the particular disease condition being treated accordingto principles known in the art.

Typically unit dosage forms will contain about 1 mg to 500 mg of acompound of this invention.

Therefore in a further aspect, the present invention provides a compoundof the formula I or a pharmaceutically acceptable salt or in vivohydrolysable ester thereof for use in a method of therapeutic treatmentof the human or animal body. In particular we disclose use in thetreatment of a disease or condition mediated by MMP13 and/or aggrecanaseand/or MMP9 and/or MMP12.

In yet a further aspect the present invention provides a method oftreating a metalloproteinase mediated disease condition which comprisesadministering to a warm-blooded animal a therapeutically effectiveamount of a compound of the formula I or a pharmaceutically acceptablesalt or in vivo hydrolysable ester thereof. Metalloproteinase mediateddisease conditions include arthritis (such as osteoarthritis),atherosclerosis, chronic obstructive pulmonary diseases (COPD).

In another aspect the present invention provides a process for preparinga compound of the formula I or a pharmaceutically acceptable salt or invivo hydrolysable ester thereof which process comprises reacting acompound of the formula II with an appropriate compound of the formulaR1CHO to yield an alkene of formula III, which is then converted to acompound of formula IV, which is a precursor to compound I, andoptionally thereafter forming a pharmaceutically acceptable salt or invivo hydrolysable ester of the compound of formula I, as set out below:

A compound of formula II is conveniently prepared by reacting a compoundof formula V with a compound of formula VI, wherein B′ is a precursor ofB and X′ represents X or a precursor of X or an activated form of Xsuitable for reaction with B′. II may also be prepared from compound VIIas shown below:

It will be appreciated that many of the relevant starting materials arecommercially available. In addition the following table shows details ofaldehyde intermediates and their corresponding registry numbers inChemical Abstracts.

Chemical Abstracts RCHO Registry Numbers3-(2-pyrimidinylthio)-propionaldehyde 155957-56-53-(2-pyrazinyl)butyraldehyde 177615-94-0 3-phenylsulphonylamido-propanal 57483-28-0 4-(4-methoxyphenyl)-butyraldehyde 160093-24-34-(3-methoxyphenyl)-butyraldehyde 113504-55-5Aldehydes without Chemical Abstracts Registry Numbers

3-(2-pyrimidyl)propionaldehyde. To a solution of 2-Bromopyrimidine (7.95g, 0.05 M) in acetonitrile (150 mL) was added propargylalcohol (4.2 g,0.075 M), bis-(triphenylphosphine)-palladium(II)chloride (750 mg, 1 mM),copper iodide (100 mg, 0.5 mM) and triethylamine (25 mL, 0.25 M) and themixture was stirred and heated at 70° C. for 2 hours. An additionalamount of propargyl alcohol (2.1 g, 0.038 M),bis-(triphenylphosphine)-palladium(II)chloride (375 mg, 0.5 mil), andcopper iodide (50 mg, 0.25 mil) was then added to the reaction mixturewhich was stirred and heated at 70° C. for an additional 1 hour.

The reaction mixture was evaporated to dryness and the residue which waspre-adsorbed on to silica, chromatographed. Elution with ethyl acetategave 3-(2-pyrimidyl)prop-2-yn-3-ol as a yellow solid 4.45 g (66%). NMR(CDCl₃) 2.9 (1H, t), 4.5 (2H, d), 7.3 (1H, d), 8.8 (2H, t), MS found MH⁺135.

3-(2-pyrimidyl)prop-2-yn-1-ol (4.45 g, 0.033 M) was dissolved in ethylacetate (140 mL), 10% Pd/C (890 mg) was added and the mixture stirredunder an atmosphere of hydrogen for 6 hours. The reaction mixture wasfiltered through Celite and the filtrate evaporated to give3-(2-pyrimidyl)propan-1-ol as a yellow oil, 4.15 g (91%). NMR (CDCl₃)2.1 (2H, m), 3.2 (2H, t), 3.8 (2H, t), 7.2 (1H, t), 8.7 (2H, d) MS foundMH⁺ 139.

3-(2-pyrimidyl)propan-1-ol was oxidized to give3-(2-pyrimidyl)propionaldehyde using the following Swern conditions. Tooxalyl chloride (14.3 ml) dissolved in dichloromethane (700 ml) wasadded DMSO (21.3 ml), maintaining the temperature below −60° C. After 15minutes the alcohol (20.8 g) dissolved in dichloromethane (20 ml) wasslowly added followed 30 minutes later by triethylamine (125 ml). After15 minutes the reaction mixture was allowed to warm to room temperaturewhen water (100 ml) was added. The solvents were separated and theorganic layer was washed with water (3×150 ml), dried (MgSO₄) andevaporated to give an oil which was purified by flash columnchromatography eluting with ethyl acetate/methanol (5%) to give theproduct (8.71 g, 43%) as an oil. NMR CDCl₃ 3.0 (2H, t), 3.4 (2H, t), 7.1(1H, t), 8.7 (2H, d), 9.9 (1H, s).

Using the procedure described above the following aldehydes wereprepared:

4-(2-pyrimidyl)butyraldehyde by using 3-butyn-1-ol in place ofpropargylalcohol NMR CDCl₃ 9.8(1H, s), 8.6 (2H, m), 7.15 (1H, m), 3.0(2H, m), 2.5 (2H, m), 2.2 (2H, m).

3-(2-pyrazinyl)propionaldehyde by using 2-bromopyrazine in place of2-bromopyrimidine NMR (d6-DMSO) 9.77 (s, 1H), 8.61 (d, 1H), 8.54 (dd,1H), 8.46 (d, 1H), 3.10 (t, 2H), 2.92 (t, 2H).

4-(2-pyrazinyl)butyraldehyde by using 2-bromopyrazine in place of2-bromopyrimidine and 3-butyn-1-ol in place of propargyl alcohol NMR(d6-DMSO) 9.68 (s, 1H), 8.56 (m, 2H), 8.49 (m, 1H), 2.80 (t, 2H), 2.5(m, 2H), 1.96 (m, 2H).

4-(4-trifluoromethylpyrimidin-2-yl)butanal by using2-chloro-4-trifluoropyrimidine [CAS registry number 33034-67-2] in placeof 2-bromopyrimidine and 3-butyno-1-ol in place of propargyl alcohol ¹HNMR (CDCl₃): 9.80 (s, 1 H), 8.92 (d, 1 H, J=5.0 Hz), 7.47 (d, 1 H, J=5.0Hz) 3.11 (dd, 2 H, J 7.5, 7.5 Hz), 2.60 (dd, 2 H, J=6.1, 6.1 Hz), 2.21(m, 3 H).

4-(5-fluoropyrimidin-2-yl)butanal by using 2-chloro-5-fluoro-pyrimidine[CAS registry number 62802-42-0] in place of 2-bromopyrimidine and3-butyno-1-ol in place of propargyl alcohol ¹H NMR (CDCl₃): 9.90 (s, 1H), 8.52 (s, 2 H, J=5.0 Hz), 7.47, 3.47 is (m, 2 H), 3.33 (dd, 2 H,J=6.8, 6.8 Hz), 3.02 (m, 2 H).

4-(4-methoxypyrimidin-2-yl)butanal by using2-chloro-4-methoxy-pyrimidine [CAS registry number 22536-63-6] in placeof 2-bromopyrimidine and 3-butyno-1-ol in place of propargyl alcohol ¹HNMR (CDCl₃): 9.80 (s, 1 H), 8.34 (d, 1 H, J=5.0 Hz), 6.55 (d, 1 H, J=5.0Hz), 3.97 (s, 3 H), 2.91 (dd, 2 H, J=6.8, 6.8 Hz), 2.58 (m, 2 H), 2.20(m, 2 H).

4-(5-ethylpyrimidin-2-yl)butanal by using 2-chloro-5-ethyl-pyrimidine[CAS registry number 111196-81-7] in place of 2-bromopyrimidine and3-butyno-1-ol in place of propargyl alcohol ¹H NMR (CDCl₃): 9.79 (s, 1H), 8.51 (s, 2 H), 2.99 (dd, 2 H, J=7.4, 7.4 Hz), 2.54 (m, 4 H), 2.17(p, 1 H, J=7.4 Hz), 1.04 (t, 2 H, J=7.2 Hz).

5-(2-pyrimidyl)pentanal by using 2-bromopyrimidine and 4-pentyn-1-ol inplace of propargul alcohol: NMR (CDCl₃) 9.8 (1H, s), 8.65 (2H, m), 7.1(1H, m), 3.0 (2H, m), 2.5 (2H, m), 1.9 (2H, m), 1.7 (2H, m).

3-(5-bromopyrimidin-2-yl)propanal by using 2-iodo-5-bromopyrimidine inplace of 2-bromopyrimidine ¹H NMR (CDCl₃): 9.90 (s, 1H), 8.70 (s, 2H),3.30 (dd, 2H), 3.0 (dd, 2H).

4-(4-pyrimidyl)-butan-1-al. 2,4-Dicloropyrimidine (4.47 g, 0.03M) wasdissolved in triethylamine (250 ml) under argon. (Ph₃P)₂PdCl₂ (420 mg,0.006M, CuI (28 mg, 0.00015M) and 3-butyn-1-ol (2.36 ml, 0.03M) wereadded and the mixture was stirred at ambient temperature for 18 hrs.After evaporation to dryness, water (250 ml), was added and extractedwith dichloromethane. The combined organic phases were dried andevaporated to dryness. The residual oil was chromatographed, elutingwith iso-hexane/ethyl acetate 1:1 to yield 4-(2chloro-4-pyrimidyl)-3-butyn-1-ol as an oil (3.3 g) NMR (CDCl₃) d, 8.5,(d, 1H); 7.3, (d, 1H); 3.9, (t, 2H); 2.8, (m, 2H); 1.6, (s, 1H). MassSpec found MH+ 183. This material was hydrogenated as described above,but in the presence of 1 equivalent of triethylamine, to give therequired saturated alcohol which was oxidised using the previouslydescribed Swem oxidation to give the required4-(4-pyrimidyl)-butan-1-al. NMR CDCl₃ d, 9.8, (s, 1H); 9.1; (s, 1H);8.5, (d, 1H); 7.1, (d, 1H); 2.8, (t, 2H); 2.5, (t, 2H); 2.1, (m, 2H).Mass spec found MH− 149.3-(5-fluoropyrimidin-2-yl)propanal. To a stirred solution of(E)-1-ethoxy-3-(5-fluoropyrimidin-2-yl)prop-2-enyl ethyl ether and(Z)-1-ethoxy-3-(5-fluoropyrimidin-2-yl)prop-2-enyl ethyl ether (9.7 g,43 mmol) in dry ethanol (100 ml) at room temperature under an atmosphereof argon, was added 10% palladium on activated charcoal (1.0 g). Thereaction flask was then evacuated and filled with hydrogen gas. Themixture was then stirred for 18 hours at room temperature. The reactionwas then filtered through a pad of celite and evaporated under reducedpressure to give a yellow oil (8.7 g, 89%). To a solution of this oil(15 g, 66 mmol) in THF (200 ml) at room temperature was added an aqueoussolution of hydrochloric acid (36 ml of a 2M solution, 72 mmol) and thereaction was stirred at room temperature for 3 hours. The reaction wasthen diluted with ethyl acetate (100 ml) and the pH of the mixturebrought to pH=9 by the addition of aqueous sodium hydrogen carbonatesolution (saturated, 100 ml). The layers were then separated and theaqueous phase extracted with ethyl acetate (3×100 ml). The combinedorganic extracts were then dried (Na₂SO₄), filtered and evaporated underreduced pressure to give 3-(5-fluoropyrimidin-2-yl)propanal (16 g) whichwas used without further purification. ¹H NMR (CDCl₃): 9.90 (s, 1 H),8.50 (s, 2 H), 3.33 (dd, 2 H, J=6.9, 6.9 Hz), 3.00 (dd, 2 H, J=6.9, 6.9Hz).

The starting material was obtained by the following method: To asolution of 2-chloro-5-fluoro-pyrimidine [CAS registry number62802-42-0] (9.0 g, 68 mmol) and1-tributylstannyl-3,3-diethoxyprop-1-ene (42.8 g, 102 mmol, 5:1 mixtureof E:Z isomers) in dry DMF (140 ml) under an atmosphere of dry argon,was added sequentially solid potassium carbonate (9.4 g, 68 mmol),tetraethylammonium chloride (11.2 g, 68 mmol) andbis(triphenylphosphine)palladium(II)chloride (2.4 g, 3.4 mmol). Theresulting mixture was then heated to 120° C. for 3 hours. The reactionwas then cooled to room temperature and was diluted with water (100 ml)and diethyl ether (150 ml). This mixture was then filtered through a padof celite. The layers were separated and the aqueous phase extractedwith diethyl ether (3×100 ml). The combined organic extracts were thendried (MgSO₄), filtered and evaporated under reduced pressure. Flashchromatography (silica gel, 10% ethyl acetate in hexanes) then gave theproduct as a pale yellow oil and a 3:1 mixture of E:Z isomers (9.7 g,63%).

E-isomer: ¹H NMR (CDCl₃): 8.53 (s, 2 H), 6.99 (dd, 1 H, J=15.4, 4.1 Hz),6.86 (d, 1 H, J=15.4 Hz), 5.14 (d, 1 H, J=4.1 Hz), 3.56 (m, 4 H), 1.24(t, 6 H, J=7.1 Hz).

Z-isomer: ¹H NMR (CDCl₃): 8.57 (s, 2 H), 6.65 (d, 1 H, J=12.1 Hz), 6.49(d, 1 H, J=7.5 Hz), 6.09 (dd, 1 H, J=12.1, 7.5 Hz), 3.70 (m, 4 H), 1.21(t, 6 H, J=7.1 Hz).

An analogous method was used to prepare the following aldehydes usingthe appropriately substituted 2-chloro-pyrimidine:

3-(4-methoxypyrimidin-2-yl)propanal ¹H NMR (CDCl₃): 9.82 (s, 1 H), 8.34(d, 1 H, J=8.4 Hz), 6.55 (d, 1 H, J=7,4 Hz), 3.91 (s, 3 H), 3.28 (dd, 2H, J=7.4, 7.4 Hz) 2.99 (dd, 2 H, J=7.4, 7.4 Hz).

3-(4-trifluoromethylpyrimidin-2-yl)propanal ¹H NMR (CDCl₃): 9.92 (s, 1H), 8.90 (d, 1 H, J=5.0 Hz), 7.47 (d, 1 H, J=5.0 Hz), 3.43 (dd, 2 H,J=6.8, 6.8 Hz).3.07 (dd, 2 H, J=6.8, 6.8 Hz).

3-(5-ethylpyrimidin-2-yl)propanal ¹H NMR (CDCl₃): 9.91 (s, 1 H), 8.49(s, 2 H), 3.31 (dd, 2 H, J=6.9, 6.9 Hz) 2.98 (dd, 2 H, J=6.9, 6.9 Hz),2.61 (q, 2 H, J=7.6 Hz), 1.26 (t, 3 H, J=7.6 Hz).

3,5,5-trimethyl-1-propanal hydantoin

A solution of 3,5,5-trimethyl hydantoin [CAS (6345-19-3)] (3.5 g, 0.025mol), 2-(2-bromoethyl)-1,3-dioxolane (4.8 ml, 0.041 mol), K₂CO₃ (8.5 g,0.062 mol), benzyltrimethylammonium chloride (2.23 g, 0.012 mol) in MeCN(100 ml) was refluxed together for 24 hrs. Allowed the reaction to coolto RT and filtered, the filtrate was evaporated in vacuo. The residuewas taken into DCM then washed with water (×3), before evaporating invacuo. The residue was azeotroped with toluene (×3) to afford a yellowoil (5.4 g). The oil was then stirred in THF (30 ml) with conc. HCl (4ml) at RT for 20 hrs. Neutralised with aqueous NaHCO3 and extracted withDCM (×8). The combined organics were dried over Na₂SO₄ and evaporated invacuo to afford a yellow oil (4.3 g) ¹H NMR (CDCl₃): 9.82 (s, 1H), 3.62(t, 2H), 3.04 (s, 3H), 2.90 (m, 2H), 1.37 (s, 6H).

1,5,5-trimethyl-3-propanal hydantoin

1,5,5-trimethylhydantoin [CAS (6851-81-6)] (5.0 g, 35.0 mol) was addedto a mixture of NaOEt (0.02 g, 0.298 mmol, catalytic) and EtOH (8 ml),and stirred under Argon. The mixture was warmed to 30° C. before addingacrolein (2.35 ml) slowly, and the reaction exotherms to 45° C. Thereaction was allowed to cool to RT and stirred for a further 2 hrs. AcOH(0.136 ml, 2.4 mmol) and silica gel (3.5 g) were added to the mixturebefore evaporating en vacuo. The product on silica was chromatagraphedon a silica column (eluant:5% acetone/DCM) to afford a clear oil (6.2g). Further purification of the residue on alumina (eluant:DCM) affordeda clear oil (2.7 g). ¹H NMR (CDCl₃): 9.78 (s, 1H), 3.88 (t, 2H), 2.86(s, 3H), 2.82 (m, 2H), 1.37 (s, 6H).

In an analogous manner 1,5,5-trimethyl-3-butanal hydantoin was prepared[M+H 213].

3-(3-chlorophenyl)butyraldehyde. A mixture of 3-chloroiodobenzene (2.38g), palladium acetate (20 mg), sodium bicarbonate (1.01 g) and crotylalcohol (1.28 ml) in N-methylpyrrolidone (4 ml) was stirred and heatedat 130° C. for 2 hours. The reaction mixture was allowed to cool, water(50 ml) was added and the mixture was extracted with diethyl ether (2×50ml). The combined organic extracts were dried and the residue obtainedon removal of the solvent was purified by chromatography through silicaeluting with a mixture of ethyl acetate and methylene chloride (1:20) togive the title compound as an oil, yield 519 mg, M−H=181

3-(2-pyridyl)butyraldehyde. Prepared by Swem oxidation of thecorresponding alcohol (CAS 90642-86-7).

3-(5-fluoropyrimidin-2-yl)butanal

Concentrated hydrochloric acid (1 m) was added to a stirring solution of2-[2-(1,3-dioxolan-2-yl)-1-methylethyl]-5-fluoropyrimidine (1.1 g) intetrahydrofuran (10 ml) at ambient temperature, stirred for 3 hours thenadded solid sodium hydrogen carbonate to neutral pH. The mixture waspoured onto a Chemelute carrtridge and washed with ethyl acetate (3×20ml), the combined organics were dried over Na₂SO₄ and evaporated invacuo to afford 3-(5-fluoropyrimidin-2-yl)butanal (300 mg, 35%) whichwas used without further purification.

The starting material was prepared as follows:

2-[2-(1,3-dioxolan-2-yl)-1-methylethyl]-5-fluoropyrimidine

To a stirring suspension of activated “Rieke” zinc in tetrahydrofuran(21 ml, 1.53M) was added 2-(2-bromopropyl)-1,3-dioxolane (6.6 g) intetrahydrofuran (50 ml), a rise in temperature from 21° C. to 40° C. wasobserved, heated at 40° C. for 1 hour then allowed to cool to ambienttemperature before adding 2-chloro-5-fluoropyrimidine (3 g) and[1,2-Bis(diphenylphosphino)-propane]dichloronickel(II)chloride (368 mg).The mixture was stirred at ambient temperature for 4 hours then filteredthrough a pad of celite and the filtrate evaporated under reducedpressure. Flash chromatography (silica gel, haxane-25% ethyl acetate inhexanes) then gave the product as a pale yellow oil (1.1 g); ¹H NMR(d6-DMSO): 8.81 (s, 2H), 4.73 (dd, 1H), 3.66–3.87 (m, 4H), 3.21–3.30 (m,1H), 2.19 (ddd, 1H), 1.83 (ddd, 1H), 1.27(d, 3H); m/z 213 (M+1).

2-(2-bromopropyl)-1,3-dioxolane

Crotonaldehyde (9.18 g, 108 mmol) was added dropwise to a stirringsolution of bromotrimethylsilane (24 g, 156 mmol) at 0° C., stirred for1 hour at 0° C. then warmed to room temperature and stirred for afurther 1 hour. Ethylene glycol (9.5 g, 156 mmol) and p-tolunesulphonicacid (100 mg) was added and the solution was heated to reflux, water wasremoved by use of Dean and Stark apparatus. On completion the mixturewas cooled to room temperature and washed with aqueous sodiumhydrogencarbonate solution (saturated, 2×50 ml).The residue was purified byvacuum distillation to give 2-(2-bromopropyl)-1,3-dioxolane (18.8 g,40–42° C. @1 mm Hg, 89%)

¹H NMR (CDCl₃): 5.05 (dd, 1H), 4.18–4.33 (m, 1H), 3.84–4.0 (m, 4H), 2.25(ddd, 1H), 2.03 (ddd, 1H), 1.75 (d, 3H).

An analogous method was used to prepare the following aldehydes usingthe appropriately substituted 2-chloro-pyrimidine and 1,3-dioxolane:

3-(5-chloropyrimidin-2-yl)propanal

¹H NMR (CDCl₃): 9.90 (s, 1H), 8.60 (s, 2H), 3.32 (dd, 2H), 3.04 (dd,2H).

3-(5-chloropyrimidin-2-yl)butanal

¹H NMR (CDCl₃): 9.85 (s, 1H), 8.60 (s, 2H), 3.65 (m, 1H), 3.14 (dd, 1H),2.75 (dd, 1H), 1.39 (d, 3H).

3-[2-(6-Chloropyrazinyl)]propanal

3-[2-(6-Chloropyrazinyl)]propanal diethyl acetal (200 mg, 0.82 mmol) wastreated with 2N hydrochloric acid (450 μl) in tetrahydrofuran (2.5 ml)at room temperature for 18 h. After adjusting the pH to 8 usingsaturated aqueous sodium bicarbonate, the reaction was extracted (×3)with ethyl acetate and the organics dried (anyhdrous sodium sulfate),filtered and concentrated in vacuo to give the title compound as a darkbrown oil (137 mg, 98%). This material was used without furtherpurification.

¹H NMR (CDCl₃) δ 9.85 (1H, s); 8.4 (2H, 2×s); 3.5 (2H, t); 3.0 (2H, t).

The starting material was obtained by the following method:

3-[2-(6-Chloropyrazinyl)]propanal diethyl acetal

3-[2-(6-Chloropyrazinyl)]propynal diethyl acetal, (5.5 g, 22.9 mmol) inethanol (55 ml) was degassed with argon and platinum (IV) oxide (52 mg,0.23 mmol) added. The reaction vessel was evacuated and an atmosphere ofhydrogen was introduced. After 2 days the reaction mixture wasconcentrated in vacuo and purified by flash chromatography, eluting witha gradient of 0–50% ethyl acetate in iso-hexane, to give3-[2-(6-Chloropyrazinyl)]propanal diethyl acetal as a pale yellow oil(1.17 g, 21%).

¹H NMR (CDCl₃) δ 8.4 (1H, s); 8.35 (1H, s); 4.5 (1H, t); 3.75–3.55 (2H,m); 3.55–3.4 (2H, m); 2.9 (2H, dd); 2.1 (2H, dd); 1.2 (6H, t).

3-[2-(6-Chloropyrazinyl)]propynal diethyl acetal

To a solution of 2,6-dichloropyrazine (1 g, 6.7 mmol) andpropionaldehyde diethyl acetal (1.1 ml, 7.4 mmol) in acetonitrile (10ml) at room temperature under an atmosphere of argon was addedbis(triphenylphosphine)palladium(II)dichloride (94 mg, 0.13 mmol) andcopper (I) iodide (51 mg, 0.27 mmol), followed by triethylamine (4.7 ml,33.6 mmol). The reaction was stirred at room temperature over night. Thesolvent was removed in vacuo and the residue purified by flashchromatography, eluting with 10–20% ethyl acetate in iso-hexane, to give3-[2-(6-Chloropyrazinyl)]propynal diethyl acetal as a yellow oil (660mg, 41%).

¹H NMR (CDCl₃) δ 8.6 (1H, s); 8.55 (1H, s); 5.5 (1H, s); 3.9–3.75 (2H,m); 3.7–3.4 (2H, m); 1.25 (6H, t). m/s (EI⁺) 241/243 (MH⁺).

An alternative process for preparing a compound of the formula I or apharmaceutically acceptable salt or in vivo hydrolysable ester thereofcomprises reacting a compound of the formula II with a compound of theformula R1COOR to yield a compound of the formula VIII, converting thisto a compound of the formula IX, converting the compound of formula IXto an alkene of formula III, which is then converted to a compound offormula IV, which is a precursor to compound I, and optionallythereafter forming a pharmaceutically acceptable salt or in vivohydrolysable ester of the compound of formula I, as set out below:

Appropriate esters of the formula R1COOR may be commercially orotherwise available or may be produced using, for example, an analogousprocedure to that described in Example 10. It will be appreciated thatit is possible to use any ester of the formula R1COOR (wherein R1 is aspreviously defined):—R may be any group including, for example, alkyl,aralkyl, heteroaryl etc.

The compounds of the invention may be evaluated for example in thefollowing assays:

Isolated Enzyme Assays

Matrix Metalloproteinase Family Including for Example MMP13

Recombinant human proMMP13 may be expressed and purified as described byKnauper et al. [V. Knauper et al., (1996) The Biochemical Journal271:1544–1550 (1996)].

The purified enzyme can be used to monitor inhibitors of activity asfollows: purified proMMP13 is activated using 1 mM amino phenyl mercuricacid (APMA), 20 hours at 21° C.; the activated MMP13 (11.25 ng perassay) is incubated for 4–5 hours at 35° C. in assay buffer (0.1MTris-HCl, pH 7.5 containing 0.1M NaCl, 20 mM CaCl2, 0.02 mM ZnCl and0.05% (w/v) Brij 35 using the synthetic substrate7-methoxycoumarin-4-yl)acetyl.Pro.Leu.Gly.Leu.N-3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl.Ala.Arg.NH₂in the presence or absence of inhibitors. Activity is determined bymeasuring the fluorescence at λex 328 nm and λem 393 nm. Percentinhibition is calculated as follows: % Inhibition is equal to the[Fluorescence_(plus inhibitor)−Fluorescence_(background)] divided by the[Fluorescence_(minus inhibitor)−Fluorescence_(background)].

A similar protocol can be used for other expressed and purified pro MMPsusing substrates and buffers conditions optimal for the particular MMP,for instance as described in C. Graham Knight et al., (1992) FEBS Lett.296(3):263–266.

Adamalysin Family Including for Example TNF Convertase

The ability of the compounds to inhibit proTNFα convertase enzyme may beassessed using a partially purified, isolated enzyme assay, the enzymebeing obtained from the membranes of THP-1 as described by K. M. Mohleret al., (1994) Nature 370:218–220. The purified enzyme activity andinhibition thereof is determined by incubating the partially purifiedenzyme in the presence or absence of test compounds using the substrate4′,5′-Dimethoxy-fluoresceinylSer.Pro.Leu.Ala.Gln.Ala.Val.Arg.Ser.Ser.Ser.Arg.Cys(4-(3-succinimid-1-yl)-fluorescein)-NH₂in assay buffer (50 mM Tris HCl, pH 7.4 containing 0.1% (w/v) TritonX-100 and 2 mM CaCl₂), at 26° C. for 18 hours. The amount of inhibitionis determined as for MMP13 except λex 490 nm and λem 530 nm were used.The substrate was synthesised as follows. The peptidic part of thesubstrate was assembled on Fmoc-NH-Rink-MBHA-polystyrene resin eithermanually or on an automated peptide synthesiser by standard methodsinvolving the use of Fmoc-amino acids andO-benzotriazol-1-yl-N,N,N′,N′-tetramnethyluronium hexafluorophosphate(HBTU) as coupling agent with at least a 4- or 5-fold excess ofFmoc-amino acid and HBTU. Ser¹ and Pro² were double-coupled. Thefollowing side chain protection strategy was employed; Ser¹(But),Gln⁵(Trityl), Arg^(8,12)(Pmc or Pbf), Ser^(9,10,11)(Trityl),Cys¹³(Trityl). Following assembly, the N-terminal Fmoc-protecting groupwas removed by treating the Fmoc-peptidyl-resin with in DMF. Theamino-peptidyl-resin so obtained was acylated by treatment for 1.5–2 hrat 70° C. with 1.5–2 equivalents of4′,5′-dimethoxy-fluorescein-4(5)-carboxylic acid [Khanna & Ullman,(1980) Anal Biochem. 108:156–161) which had been preactivated withdiisopropylcarbodiimide and 1-hydroxybenzotriazole in DMF]. Thedimethoxyfluoresceinyl-peptide was then simultaneously deprotected andcleaved from the resin by treatment with trifluoroacetic acid containing5% each of water and triethylsilane. The dimethoxyfluoresceinyl-peptidewas isolated by evaporation, trituration with diethyl ether andfiltration. The isolated peptide was reacted with4-(N-maleimido)-fluorescein in DMF containing diisopropylethylamine, theproduct purified by RP-HPLC and finally isolated by freeze-drying fromaqueous acetic acid. The product was characterised by MALDI-TOF MS andamino acid analysis.

Natural Substrates

The activity of the compounds of the invention as inhibitors of aggrecandegradation may be assayed using methods for example based on thedisclosures of E. C. Arner et al., (1998) Osteoarthritis and Cartilage6:214–228; (1999) Journal of Biological Chemistry, 274 (10), 6594–6601and the antibodies described therein. The potency of compounds to act asinhibitors against collagenases can be determined as described by T.Cawston and A. Barrett (1979) Anal. Biochem. 99:340–345.

Inhibition of Mettalloproteinase Activity in Cell/Tissue Based Activity

Test as an Agent to Inhibit Membrane Sheddases such as TNF Convertase

The ability of the compounds of this invention to inhibit the cellularprocessing of TNFα production may be assessed in THP-1 cells using anELISA to detect released TNF essentially as described K. M. Mohler etal., (1994) Nature 370:218–220. In a similar fashion the processing orshedding of other membrane molecules such as those described in N. M.Hooper et al., (1997) Biochem. J. 321:265–279 may be tested usingappropriate cell lines and with suitable antibodies to detect the shedprotein.

Test as an Agent to Inhibit Cell Based Invasion

The ability of the compound of this invention to inhibit the migrationof cells in an invasion assay may be determined as described in A.Albini et al., (1987) Cancer Research 47:3239–3245.

Test as an Agent to Inhibit Whole Blood TNF Sheddase Activity

The ability of the compounds of this invention to inhibit TNFαproduction is assessed in a human whole blood assay where LPS is used tostimulate the release of TNFα. Heparinized (10 Units/ml) human bloodobtained from volunteers is diluted 1:5 with medium(RPMI1640+bicarbonate, penicillin, streptomycin and glutamine) andincubated (160 μl) with 20 μl of test compound (triplicates), in DMSO orappropriate vehicle, for 30 min at 37° C. in a humidified (5% CO₂/95%air) incubator, prior to addition of 20 μl LPS (E. coli. 0111:B4; finalconcentration 10 μg/ml). Each assay includes controls of diluted bloodincubated with medium alone (6 wells/plate) or a known TNFα inhibitor asstandard. The plates are then incubated for 6 hours at 37° C.(humidified incubator), centrifuged (2000 rpm for 10 min; 4° C.), plasmaharvested (50–100 μl) and stored in 96 well plates at −70° C. beforesubsequent analysis for TNFα concentration by ELISA.

Test as an Agent to Inhibit in vitro Cartilage Degradation

The ability of the compounds of this invention to inhibit thedegradation of the aggrecan or colfagen components of cartilage can beassessed essentially as described by K. M. Bottomley et al., (1997)Biochem J. 323:483–488.

Pharmacodynamic Test

To evaluate the clearance properties and bioavailability of thecompounds of this invention an ex vivo pharmacodynamic test is employedwhich utilises the synthetic substrate assays above or alternativelyHPLC or Mass spectrometric analysis. This is a generic test which can beused to estimate the clearance rate of compounds across a range ofspecies. Animals (e.g. rats, marmosets) are dosed iv or po with asoluble formulation of compound (such as 20% w/v DMSO, 60% w/v PEG400)and at subsequent time points (e.g. 5, 15, 30, 60, 120, 240, 480, 720,1220 mins) the blood samples are taken from an appropriate vessel into10 U heparin. Plasma fractions are obtained following centrifugation andthe plasma proteins precipitated with acetonitrile (80% w/v finalconcentration). After 30 mins at −20° C. the plasma proteins aresedimented by centrifugation and the supernatant fraction is evaporatedto dryness using a Savant speed vac. The sediment is reconstituted inassay buffer and subsequently analysed for compound content using thesynthetic substrate assay. Briefly, a compound concentration-responsecurve is constructed for the compound undergoing evaluation. Serialdilutions of the reconstituted plasma extracts are assessed for activityand the amount of compound present in the original plasma sample iscalculated using the concentration-response curve taking into accountthe total plasma dilution factor.

In Vivo Assessment

Test as an Anti-TNF Agent

The ability of the compounds of this invention as ex vivo TNFαinhibitors is assessed in the rat. Briefly, groups of male WistarAlderley Park (AP) rats (180–210 g) are dosed with compound (6 rats) ordrug vehicle (10 rats) by the appropriate route e.g. peroral (p.o.),intraperitoneal (i.p.), subcutaneous (s.c.). Ninety minutes later ratsare sacrificed using a rising concentration of CO₂ and bled out via theposterior vena cavae into 5 Units of sodium heparin/ml blood. Bloodsamples are immediately placed on ice and centrifuged at 2000 rpm for 10min at 4° C. and the harvested plasmas frozen at −20° C. for subsequentassay of their effect on TNFα production by LPS-stimulated human blood.The rat plasma samples are thawed and 175 μl of each sample are added toa set format pattern in a 96 U well plate. Fifty μl of heparinized humanblood is then added to each well, mixed and the plate is incubated for30 min at 37° C. (humidified incubator). LPS (25 μl; final concentration10 μg/ml) is added to the wells and incubation continued for a further5.5 hours. Control wells are incubated with 25 μl of medium alone.Plates are then centrifuged for 10 min at 2000 rpm and 200 μl of thesupernatants are transferred to a 96 well plate and frozen at −20° C.for subsequent analysis of TNF concentration by ELISA.

Data analysis by dedicated software calculates for each compound/dose:Percent inhibition of TNFα=Mean TNFα (Controls)−Mean TNFα (Treated)×100Mean TNFα (Controls)Test as an Anti-Arthritic Agent

Activity of a compound as an anti-arthritic is tested in thecollagen-induced arthritis (CIA) as defined by D. E. Trentham et al.,(1977) J. Exp. Med. 146,:857. In this model acid soluble native type IIcollagen causes polyarthritis in rats when administered in Freundsincomplete adjuvant. Similar conditions can be used to induce arthritisin mice and primates.

Test as an Anti-Cancer Agent

Activity of a compound as an anti-cancer agent may be assessedessentially as described in I. J. Fidler (1978) Methods in CancerResearch 15:399–439, using for example the B16 cell line (described inB. Hibner et al., Abstract 283 p75 10th NCI-EORTC Symposium, AmsterdamJun. 16–19, 1998).

The invention will now be illustrated but not limited by the followingExamples:

EXAMPLE 1N-[1-([4-(4-bromophenyl)piperazino]sulfonylmethyl)-4-pyrimidin-2-ylbutyl]-N-hydroxyformamide

To a stirred solution ofN-[1-([4-(4-bromophenyl)piperazino]sulfonylmethyl)-4-pyrimidin-2-ylbutyl]hydroxylamine(497 mg, 1.0 mmol) in THF (5.0 ml) and formic acid (2.5 ml), cooled to0° C., was added a preformed mixture of acetic anhydride (566 μl, 6.0mmol) and formic acid (2.0 ml). The mixture was stirred at 0° C. for 1hour and allowed to come to room temperature. The solvents were removedby rotary evaporation and the residue purified by chromatography (50 gSilica Bond Elute, eluent 0→15% Methanol/Dichloromethane), purefractions evaporated, and crystallised from hot ethyl acetate to giveN-[1-([4-(4-bromophenyl)piperazino]sulfonylmethyl)-4-pyrimidin-2-ylbutyl]-N-hydroxyformamideas a white crystalline powder (262 mg, 51%).

NMR (300 MHz DMSO-d₆) δ/ppm: 9.87 (s, 1H*), 9.55 (s, 1H*), 8.70 (m, 2H),8.29 (s, 1H*), 7.98 (s, 1H*), 7.33 (m, 3H), 6.92 (dd, 2H), 4.68 (m,1H*), 4.13 (m, 1H*), 3.55–3.31 (m, 5H, partially obscured), 3.25–3.09(m, 7H, partially obscured), 1.80–1.50 (m, 4H). MS: ES⁺, (M+H)⁺=512, 514(Br Isotope Pattern). * rotameric signals

The starting material was prepared as follows:

i) To a solution of 1-(4-bromophenyl)piperazine hydrochloride (5.09 g,18.3 mmol) and triethylamine (7.67 ml) in dichloromethane (100 ml) wasadded methanesulfonyl chloride (2.83 ml, 36.3 mmol) dropwise. Themixture was stirred for 1 hour at room temperature then dichloromethane(100 ml) was added. The organics were washed with water (2×), brine anddried (Na₂SO₄) and evaporated in vacuo to a yellow solid whichcrystallised from Ethanol and washed with diethyl ether to give1-(4-bromophenyl)-4-(methanesulfonyl)piperazine (4.74 g, 81% yield) as awhite fluffy powder.

¹H NMR (300 MHz CDCl₃) δ/ppm: 7.38 (d, 2H), 6.91 (d, 2H), 3.21 (m, 8H),2.89 (s, 3H). MS: ES+, (M+H)⁺=318, 320 (Br isotope pattern).

ii) To the 1-(4-bromophenyl)-4-(methanesulfonyl)piperazine (902 mg, 2.0mmol) suspended in anhydrous THF (15 ml), under Nitrogen, cooled tobetween −20 and −30° C. was added sequentially Lithiumbis(trimethylsilyl)amide (1.0M in THF, 4.0 ml), Chlorotrimethylsilane(217 mg, 2.0 mmol, 253 μl) and 4-pyrimidin-2-ylbutanal (300 mg, 2.0mmol). The mixture was stirred at −20° C. for 1 hour, quenched withsaturated ammonium chloride solution and allowed to stand at ambienttemperature overnight. The solvents were removed in vacuo and theresidue partitioned between dichloromethane (15 ml) and water (5 ml),the organics separated and chromatogrammed (50 g Silica Bond Elute,eluted with 0→100% Ethyl Acetate/Hexane gradient) to give the2-(5-[4-(4-bromophenyl)piperazino]sulfonylpent-4-enyl)pyrimidine as awhite crystalline material (759 mg, 84% Yield)

MS: ES⁺, (M+H)⁺=451, 453 (Br isotope pattern).

iii) To a stirred solution of the2-((E)-5-[4-(4-bromophenyl)piperazino]sulfonylpent-4-enyl)pyrimidine(451 mg, 1.0 mmol) in THF (10 ml) was added Hydroxylamine (50% solutionin water, 500 μl) and the mixture stirred overnight. The solvents wereremoved in vacuo, azeotroping with toluene (3×) to give theN-[1-([4-(4-bromophenyl)piperazino]sulfonylmethyl)-4-pyrimidin-2-ylbutyl]hydroxylamine(497 mg, quantitative)

MS: ES⁺, (M+H)⁺=484, 486 (Br isotope pattern).

EXAMPLE 2N-[1-([4-(5-chloropyridin-2-yl)piperazino]sulfonylmethyl)-3-(5-fluoropyrimidin-2-yl)propyl]-N-hydroxyformamide

Acetic anhydride (0.51 ml) was added directly to formic acid (2.0 ml)which had been cooled to 0° C. and then added a solution of2-[4-[4-(5-chloropyridin-2-yl)piperazino]sulfonyl-3-(hydroxyamino)butyl]-5-fluoropyrimidine(0.485 g) in tetrahydrofuran (11 ml). The solution was stirred at roomtemperature for 3 hours and then evaporated in vacuo, the resultingresidue was azeotroped with toluene and then it was dissolved inmethanol and heated to 40° C. for 30 minutes. The solution wasevaporated to dryness and then added diethylether and stirred at roomtemperature for 10 minutes, solid filtered, dried in vacuo to giveN-[1-([4-(5-chloropyridin-2-yl)piperazino]sulfonylmethyl)-3-(5-fluoropyrimidin-2-yl)propyl]-N-hydroxyformamide,(0218 g), mp 154–155° C.

NMR (d6-DMSO 373° K): 2.20 (m, 2H), 2.95 (m, 2H), 3.23 (dd, 1H), 3.30(m, 4H), 3.49 (dd, 1H), 3.60 (m, 4H), 4.42 (vbs, 1H), 6.88 (d, 1H), 7.59(dd, 1H), 8.05 (vbs, 1H), 8.12 (dd, 1H), 8.71 (s, 2H), 9.40 (vbs, 1H);m/z 473 (M+1).

The starting material was prepared as follows:

(i) 1-(5-chloropyridin-2-yl)-4-(methylsulfonyl)piperazine (0.600 g) wasstirred in anhydrous tetrahydrofuran (22 ml) under Argon then cooled to−10° C. before the addition of lithium bis(trimethylsilyl)amide (4.8 mlof a 1.0M solution in tetrahydrofuran). The mixture was stirred at −10°C. for 30 minutes and a solution of diethylchlorophosphate (0.345 ml)was added. The mixture was stirred at −10° C. for 15 minutes and then3-(5-fluoropyrimidin-2-yl)propanal (0.334 g) was added, stirred at −10°C. for a further 30 minutes. The mixture was allowed to warm to roomtemperature and then was washed with aqueous ammonium chloride andextracted with ethyl acetate. The organic layers were dried over Na₂SO₄.

Purification of the residue on silica eluting with 70% ethyl acetate 30%hexane afforded a 6:4 mixture of2-((E)-4-[4-(5-chloropyridin-2-yl)piperazino]sulfonylbut-3-enyl)-5-fluoropyrimidineand2-((Z)-4-[4-(5-chloropyridin-2-yl)piperazino]sulfonylbut-3-enyl)-5-fluoropyrimidine(0.44 g).

¹H NMR (CDCl₃): 8.55 (d, 1H), 8.48, (s, 1H), 7.46, (dd, 1H), 6.85, (m,1H), 6.60, (d, 1H), *6.45, (m, 1H), 6.15, (d, 1H), *6.03, (d, 1H), 3.61,(m, 4H), 3.28, (m, 2H), 3.15, (m, 4H), *2.81, (m, 2H); MS (ES+):412.3(MH⁺). * Denotes minor isomer.

(ii) To a solution of2-((E)-4-[4-(5-chloropyridin-2-yl)piperazino]sulfonylbut-3-enyl)-5-fluoropyrimidineand2-((Z)-4-[4-(5-chloropyridin-2-yl)piperazino]sulfonylbut-3-enyl)-5-fluoropyrimidine(0.44 g), in tetrahydrofuran (5 ml), was added hydroxylamine (1.0 ml,50% aqueous solution). The mixture was stirred for 18 hours and thendiluted with EtOAc(10 ml) and washed with saturated ammonium chloridesolution(10 ml. The organic layer was dried over Na₂SO₄ and evaporatedin vacuo to give2-[4-[4-(5-chloropyridin-2-yl)piperazino]sulfonyl-3-(hydroxyamino)butyl]-5-fluoropyrimidine(0.483g).

¹H NMR (CDCl₃): 8.45 (s, 2H), 8.08 (d, 1H), 7.39 (dd, 1H), 6.55 (d, 1H),5.76 (bs, 2H), 3.59 (m, 4H), 3.46 (m, 1H), 3.42 (m, 2H), 3.33 (m, 4H),3.10 (m, 4H), 2.82 (m, 1H), 2.15 (m, 1H), 2.01 (m, 1H); MS (ES+):445.3(MH⁺).

EXAMPLE 3

The following compounds were prepared

Prepared using method in B X R1 mpt M + H Example 5-Cl-2-Pyridyl N1,5,5-trimethyl-3-hydantoinCH2CH2 517.3 2 (5-Cl-2-pyridyl)oxy C4-Cl-phenyl 474.3 1 5-Cl-2-Pyridyl N 3,5,5-trimethyl-1-hydantoinCH2CH2517.3 1 (5-Cl-2-pyridyl)oxy C 2-PyrimidinylCH2CH2 470.3 1 5-Cl-2-PyridylN 2-pyrimidine-SCH2CH2 487 1 (5-Br-2-pyridyl)oxy C2-PyrimidinylCH2CH2CH2 528.2 1 5-Cl-2-Pyridyl N 3-(OCH2Ph)—Ph 531 13,4-diCl-phenyl N 2-PyrimidinylCH2CH2CH2 502 1 4-Cl-phenyl N2-PyrimidinylCH2CH2CH2 468 1 5-Cl-2-Pyridyl N 3-CF3—Ph 493 1 4-Cl-phenylN 3-Pyridyl 397.4 2 5-Cl-2-Pyridyl N 4-CF3—Ph 493 1 5-Cl-2-Pyridyl N3-Thiophenyl 431 1 5-Cl-2-Pyridyl N 2-PyrazinylCH2CH2CH2 469 25-Cl-2-Pyridyl N 2-PyrazinylCH2CH2 455.4 2 3-Cl-phenyl N2-PyrimidinylCH2CH2CH2 468.4 2 6-Me-4-pyrimidinyl N2-PyrimidinylCH2CH2CH2 450.5 2 5-cyano-2-pyridyl N 2-PyrazinylCH2CH2CH2460.5 2 5-cyano-2-pyridyl N 2-PyrazinylCH2CH2 446.5 2 4-F—Ph N2-PyrimidinylCH2CH2 438 1 5-CF3-2-Pyridyl N 2-PyrimidinylCH2CH2 489 15-cyano-2-pyridyl N 2-PyrimidinylCH2CH2 446 1 5-CF3-2-Pyridyl N2-PyrimidinylCH2CH2CH2 503 1 5-Cl-2-Pyridyl N 4-PyrimidinylCH2CH2CH2 4691 4-F—Ph C 2-PyrimidinylCH2CH2CH2 451 2 4-F—Ph C 2-PyrimidinylCH2CH2 4372 5-Cl-2-Pyridyl N 2-(4-MeO-Pyrimidinyl)CH2CH2 485 2 5-Cl-2-Pyridyl C2-PyrimidinylCH2CH2CH2 468 2 5-Cl-2-Pyridyl C 2-PyrimidinylCH2CH2 454 25-Cl-2-Pyridyl N 2-(4-CF3-Pyrimidinyl)-CH2CH2 523 2 5-Cl-2-Pyridyl N2-(5-Ethyl-Pyrimidinyl)CH2CH2 483 2 5-Cl-2-Pyridyl N2-(4-MeO-Pyrimidinyl)CH2CH2CH2 499 2 5-cyano-2-pyridyl N2-(4-MeO-Pyrimidinyl)CH2CH2CH2 490 2 5-Cl-2-Pyridyl N2-(5-F-Pyrimidinyl)CH2CH2CH2 487 2 5-Br-2-Pyridyl N2-(4CF3-Pyrimidinyl)CH2CH2CH2 583 2 5-Cl-2-Pyridyl N2-(4CF3-Pyrimidinyl)CH2CH2CH2 537 2 5-cyano-2-pyridyl N2-(4CF3-Pyrimidinyl)CH2CH2CH2 528 2 5-Cl-2-Pyridyl N2-(5-Ethyl-Pyrimidinyl)CH2CH2CH2 497 2 5-Br-2-Pyridyl N2-(5-Ethyl-Pyrimidinyl)CH2CH2CH2 541/543 2 5-cyano-2-pyridyl N2-(5-Ethyl-Pyrimidinyl)CH2CH2CH2 488 2 4-F—Ph N PhSO2NHCH2CH2 515 15-Cl-2-Pyridyl C PhCH(Me)CH2 64–65 2 4-F—Ph N1,5,5-trimethyl-3-hydantoinCH(Me)CH2 85 1 4-F—Ph N 4-MeO—PhCH(Me)CH2 4801 4-F—Ph N 3-MeO—PhCH(Me)CH2 480 1 4-F—Ph C1,5,5-trimethyl-3-hydantoinCH(Me)CH2 77–79 1 4-Cl—Ph N 3-Cl—PhCH(Me)CH2500, 502 1 6-Cl-2-pyrimidinyl N 2-pyrazinylCH(Me)CH2 79–81 470 15-Cl-2-Pyridyl N 2-pyridylCH(Me)CH2 468 2 5-cyano-2-pyridyl N2-pyridylCH(Me)CH2 459 2 5-cyano-2-pyridyl N 2-pyrazinylCH(Me)CH2 80 4602 5-CN-2-Pyridyl N 2-PyrimidinylCH2CH2CH2CH2 474.5 1 4-Cl-Phenyl N2-PyrimidinylCH2CH2CH2CH2 482.45 1 5-Cl-2-Pyridyl N2-PyrimidinylCH2CH2CH2CH2 483.4 1 5-Cl-2-Pyridyl N 4-Cl-Phenyl 459.3 15-F-2-Pyridyl N 2-PyrimidinylCH2CH2CH2 453.2 2 5-F-2-Pyridyl N2-(5-F-Pyrimidinyl)CH2CH2 457.1 2 5-Br-2-Pyridyl N2-(5-F-Pyrimidinyl)CH2CH2 517/519 2 4-Cl-Phenyl N2-(5-F-Pyrimidinyl)CH2CH2 472.1 2 5-CN-2-Pyridyl N2-(5-F-Pyrimidinyl)CH2CH2 464.18 2 5-CF3-2-Pyridyl N2-(5-F-Pyrimidinyl)CH2CH2 507.14 2 5-Cl-2-Pyridyl N2-(5-Br-Pyrimidinyl)CH2CH2 533/535 2 5-F-2-Pyridyl N2-(5-Br-Pyrimidinyl)CH2CH2 517/519 2 4-F-Phenyl N2-(5-Br-Pyrimidinyl)CH2CH2 516/518 2 5-F-2-Pyridyl N2-(5-Me-Pyrimidinyl)CH2CH2 453.5 2 4-Cl-Phenyl N2-(5-Me-Pyrimdiinyl)CH2CH2 468.4 1 5-Br-2-Pyridyl N2-(5-Me-Pyrimidinyl)CH2CH2 513/515 2 5-CF3-2-Pyridyl N2-(5-Me-Pyrimidinyl)CH2CH2 503.4 2 5-F-2-Pyridyl N2-(4-CF3-Pyrimidinyl)CH2CH2 507.06 2 4-Cl-Phenyl N2-(4-CF3-Pyrimidinyl)CH2CH2 521.9 2 5-CF3-2-Pyridyl N2-(4-CF3-Pyrimidinyl)CH2CH2 556.95 2 5-Br-2-Pyridyl N2-(4-CF3-Pyrimidinyl)CH2CH2 566/568 2 5-Cl-2-PYridyl N2-(5-Cl-Pyrimidinyl)CH2CH2 489/491 2 5-Br-2-Pyridyl N2-(5-Cl-Pyrimidinyl)CH2CH2 532/534 2 5-F-2-Pyridyl N2-(5-Cl-Pyrimidinyl)CH2CH2 473 2 4-F-Phenyl N 2-(5-Cl-Pyrimdinyl)CH2CH2472 2 4-Cl-Phenyl N 2-(5-Cl-Pyrimidinyl)CH2CH2 488/490 2 4-Br-2-PyridylN 2-(5-Br-Pyrimidinyl)CH2CH2 576/578/580 2 4-Cl-Phenyl N2-(5-Br-Pyrimidinyl)CH2CH2 531/533/535 2 5-CN-2-Pyridyl N3-(5-Pyridyl)CH2CH2 479/481 2 4-CF3-Phenyl N 2-PyrimidinylCH2CH2CH2 5022 4-Br-Phenyl N 2-(5-F-Pyrimidinyl)CH2CH2 518.3 2 3,4-DiCl-Phenyl N2-(5-F-Pyrimidinyl)CH2CH2 506.34 2 3-Cl-Phenyl N2-(5-F-Pyrimidinyl)CH2CH2 472.38 2 4-CF3-Phenyl N2-(5-F-Pyrimidinyl)CH2CH2 506.4 2 4-F—Ph N 2-PyrimidinylCH2CH2 87–89 13,4-di-Cl—Ph N 2-PyrimidinylCH2CH2 489 1 4-Cl—Ph N 2-PyrimidinylCH2CH2455 1 5-Me-2-Pyridyl N 2-PyrimidinylCH2CH2CH2 449 1 5-Me-2-Pyridyl N2-PyrimidinylCH2CH2 435 1 4-F—Ph N 2-PyrazinylCH2CH2CH2 452 1 4-F—Ph N(6-Cl-2-pyrazinyl)CH2CH2 91–92 2 4-F—Ph N 5-F-2-PyrimidinylCH(CH3)CH2143–4 2 4-Cl—Ph N 2-PyrazinylCH(CH3)CH2 468 1 4-F—Ph C5-F-2-PyrimidinylCH(CH3)CH2 469 1

The starting piperazine and piperidine sulphonamides required for thesynthesis of compounds were available commercially or were prepared asshown below:

1-(4-fluorophenyl)-4-(methanesulfonyl)piperazine

To a solution of 1-(4-fluorophenyl)piperazine (35 g, 194 mmol) andpyridine (17.5 ml) in dry dichloromethane (200 ml) at 0° C. was addedmethanesulfonyl chloride (20 ml, 258 mmol) dropwise. The mixture wasstirred for 3 hours at room temperature. The mixture was washed withwater and extracted with dichloromethane (2×100 ml). The organic layerswere dried with MgSO₄ and evaporated in vacuo. The residue wastriturated and washed with methanol to give1-(4-fluorophenyl)-4-(methanesulfonyl)piperazine (39.35 g) as whitecrystals.

¹H NMR (CDCl₃): 7.00 (m, 2H), 6.90 (m, 2H), 3.40 (m, 4H), 3.20 (m, 4H),2.83 (s, 3H).

The aryl/heteroarylpiperazines and piperidines used as startingmaterials were commercially available or are described in the scientificliterature.

1-(6-chloropyrimidin-4-yl)-4-mesylpiperazine

A mixture of 4,6-dichloropyrimidine (39.4 g), 1-mesylpiperazinehydrochloride (55.7 g) and triethylamine (116 ml) in ethanol (500 ml)was stirred at reflux temperature for 4 hours. The mixture was thenstirred at room temperature for 12 hours. The solid, which hadseparated, was collected by filtration, slurry washed with ethanol (2×80ml, 160 ml) then with diethyl ether (150 ml), and dried to give1-(6-chloropyrimidin-4-yl)-4-mesylpiperazine as a cream solid (71.9 g).mp 200–202° C.

NMR (d6-DMSO): 2.88 (s, 3H), 3.18 (m, 4H), 3.80 (m, 4H), 7.04 (s, 1H),8.38 (m, 1H); m/z 277.3 (M+1).

Using an analogous procedure 1-mesylpiperazine hydrochloride,CAS(161357-89-7), was reacted with the appropriate chloropyridine togive the following, compounds.

R m/z (M + 1) 5-Cl-2-pyridyl 276 5-CF₃-2-pyridyl 310 5-CN-2-pyridyl 2675-Br-2-pyridyl 320/3222-(4-piperidinyloxy)-5-chloropyridine

i) NaH (2.88 g, 66 mmol, 55% dispersion in mineral oil) was stirred indry DME (200 ml), under Argon. A mixture of 2,5-dichloropyridine (8.87g, 60 mmol) and 4-hydroxypiperidine (6.67 g, 66 mmol) dissolved in dryDME (200 ml) was added to the NaH suspension dropwise, over a period of30 minutes. After complete addition the reaction is heated to 82° C. for48 hrs, maintaining the Argon blanket. The reaction was slowly quenchedwith water before removing most of the THF. Extracted the aqueous withDCM (×3). The organic layers were dried with Na₂SO₄ and evaporated invacuo to afford 2-(4-piperidinyloxy)-5-chloropyridine as a yellow oil(12.7 g, quantitative).

¹H NMR (DMSO): 8.17 (d, 1H), 7.76 (dd, 1H), 6.81 (d, 1H), 4.96 (m, 1H),2.93 (m, 2H), 2.53 (m, 2H), 1.91 (m, 2H), 1.46 (m, 2H); MS (ES+): 213.3(MH⁺), 225.3 (MNa⁺).

In an analogous manner 2-(4-piperidinyloxy)-5-bromopyridine was preparedMH+257.3

EXAMPLE 4 ResolutionN-[(1S)-1-({[4-(5-chloropyridin-2-yl)piperazino]sulfonyl}methyl)-4-(pyrimidin-2-yl)butyl]-N-hydroxyformamide

To the carbamate 1 (3.8 g, 5.66 mmol) dissolved in THF (76 ml) was addedmethanol (76 ml), followed by water (38 ml), and to this solution wasadded lithium hydroxide monohydrate (2.37 g, 56.6 mmol). After stirringfor 2 hours at room temperature the solvents were removed under reducedpressure and the residue dissolved in water (250 ml), washed with ethylacetate (200 ml) and diethylether (2×250 ml). Saturated aqueous ammoniumchloride was added until the aqueous layer was approximately pH 8 and itwas then extracted with dichloromethane (3×250 ml). The combineddichloromethane extracts were dried (MgSO₄) and evaporated to give theproduct as a white powder (2.2 g, 83%). Chiral HPLC using a Chiralpak AScolumn showed the product had been isolated in 96% ee (believed to haveS stereochemistry). Mpt (from EtOH) 124.5–126.5° C.;

[a]_(D) ²⁵=−17.2 (MeOH); NMR CDCl₃ d 9.9 (br s, 1H)*; 8.7 (m, 2H); 8.5(s, 1H)*; 8.1 (br s, 1H); 8.0 (s, 1H)*; 7.5 (dd, 1H); 7.2 (m, 1H); 6.6(d, 1H); 4.9 (m, 1H)*; 4.2 (m, 1H)*; 3.7–3.5 (m, 4H); 3.5 (m, 1H)*;3.4–3.2 (m, 4H); 3.3 (m, 1H)*; 3.1–2.9 (m, 3H); 2.0–1.6 (m, 4H). MS forC₁₉H₂₅ClN₆O₄S (M+H) calcd 469. found 469. * rotameric signals

To the reverse hydroxamate 2 (18.76 g, 40 mmol) dissolved indichloromethane (300 ml) and cooled to 0° C. was added triethylamine(10.4 ml, 75mmol) followed by (4S)-4-Benzyl-2-oxazolidinone-3-carbonylchloride (10.55 g, 44 mmol)[CAS number 139149-49-8]. After stirring for3 hours at −3–0° C. the mixture was washed with water (250 ml), dried(MgSO4), and evaporated to give a beige foam (27.1 g). The diastereomerswere separated using preparative hplc eluting with ethyl acetate/EtOH(5%). The more polar diastereomer was isolated in 35% yield. MS forC₃₀H₃₄ClN₇O₇S (M+H) calcd 672. found 672.

Compound 2 was prepared using the methods given in Example 2: (M+H 469),mpt 131–134° C.; NMR (DMSO) 9.8 (1H, br), 8.7 (2H, m), 8.3 and 7.9 (1H,s), 8.1 (2H, s), 7.6 (1H, m), 6.9(1H, m), 4.1 (1H, br m), 3.6 (4H, m),3.2 (6H, m), 2.8 (2H, m), 1.8 (4H, m).

EXAMPLE 5

In an analogous manner to that given in Example 4 the followingcompounds were produced:

N-[(1S)-1-({[4-(5-bromopyridin-2-yl)piperazino]sulfonyl}methyl)-4-(pyridin-2-yl)butyl]-N-hydroxyformamide

NMR CDCl₃ d 11.9 (br s, 1H)*; 8.5 (s, 1H)*; 8.5–8.4 (m, 1H); 8.2 (m,1H); 8.1 (s, 1H)*; 7.8–7.7 (m, 1H); 7.6 (m, 1H); 7.3–7.2 (m, 2H); 6.6(m, 1H); 5.0–4.9 (m, 1H)*; 4.3–4.2 (m, 1H)*; 3.7–3.6 (m, 4H); 3.6 (m,1H)*; 3.4–3.3 (m, 4H); 3.3 (m, 1H)*; 3.1 (dd, 1H)*, 2.9 (m, 1H)*,2.9–2.8 (m, 2H); 2.1–1.6 (m, 4H). MS for C₂₀H₂₆BrN₅O₄S (M+H) calcd 514.found 514. [a]_(D) ²⁵=−14 (c=2.3, MeOH) * rotameric signals

The racemic starting material was prepared using the method given inExample 2. M+H=512/514.

N-[(1S-1-({[4-(5-chloropyridin-2-yl)piperazino]sulfonyl}methyl)-3-(5-fluoropyrimidin-2-yl)propyl]-N-hydroxyformamide

¹H NMR (DMSO, 373K): 9.44 (br s, 1 H), 8.70 (s, 2 H), 8.10 (d, 1 H,J=2.6 Hz), 8.05 (br s, 1 H), 7.57 (dd, 1 H, J=9.1, 2.6 Hz), 6.86 (d, 1H, J=9.1 Hz), 4.40 (br s, 1 H), 3.59 (dd, 4 H, J=5.3, 5.0 Hz), 3.47 (dd,1 H, J=14.6, 7.4 Hz), 3.28 (dd, 4 H, J=5.3, 5.0 Hz), 3.24 (dd, 1 H,J=14.6, 4.3 Hz), 2.93 (m, 2 H), 2.16 (m, 2 H). MS (ESI): 473 (MH⁺).a_(d)=−11.03 (MeOH, c=1.242).

The racemic starting material was prepared in Example 2.

N-[(1S)-1-({[4-(4-fluorophenyl)piperazino]sulfonyl}methyl)-4-(pyrimidin-2-yl)butyl]-N-hydroxyformamide

M+H 452.44; NMR CDCl₃ d 9.9 (br s, 1H)*; 8.7 (m, 2H); 8.5 (s, 1H)*; 8.05(s, 1H)*; 7.2 (m, 1H); 7.0–6.9 (m, 4H); 4.9 (m, 1H)*; 4.2 (m, 1H)*;3.5–3.4 (m, 4H); 3.5 (m, 1H)*; 3.2–3.1 (m, 4H); 3.3 (m, 1H)*; 3.1–2.9(m, 3H); 2.0–1.6 (m, 4H). * rotameric signals

The racemic starting material was prepared using the method given inExample 3. NMR (DMSO) 10.0 (1H, br s), 8.6 (2H, m), 8.2 (1H, d), 7.2(1H, m), 6.9 (4H, m), 4.9 and 4,2 (1H, br), 3.4 (6H, m), 3.0 (6H, m),1.9 (4H, m).

EXAMPLE 6 Chromatographic ResolutionN-[(1S)-1-({[4-(5-chloropyridin-2-yl)piperazino]sulfonyl}methyl)-3-(pyrimidin-2-yl)propyl]-N-hydroxyformamideandN-[(1R)-1-({[4-(5-chloropyridin-2-yl)piperazino]sulfonyl}methyl)-3-(pyrimidin-2-yl)propyl]-N-hydroxyformamide

N-[1-({[4-(5-chloropyridin-2-yl)piperazino]sulfonyl}methyl)-3-(pyrimidin-2-yl)propyl]-N-hydroxyformamideprepared in a racemic form was separated into single enantiomer forms bychromatographic separation on a column packed with Chiralpak AD No.AD00CJ-HK002 and eluted with ethanol. Biological activity lies in thecompound eluted second from the column—assumed to have Sstereochemistry.

1^(st) enantiomer eluted MH+455.

2^(nd) enantiomer eluted MH+455.

The racemic starting material was prepared using the method given inExample 2.

MH+=455. NMR (DMSO) 9.9, 9.6 (1H br s), 8.6 (2H, m), 8.3 and 7.9 (1H,s), 8.1 (1H, dd), 7.3 (1H, m), 6.9 (1H, d), 4.7 and 4.2 (1H, broad m),3.6 (4H, m), 3.4–3.2 (6H, m), 2.8 (2H, m), 2.1 (2H, m).

EXAMPLE 7 Further Examples of Chromatographic Resolution

The following compounds were resolved using the conditions given inExample 6:

N-[(1S)-1-({[4-(5-trifluoromethylpyridin-2-yl)piperazino]sulfonyl}methyl)-3-(pyrimidin-2-yl)propyl]-N-hydroxyformamideandN-[(1R)-1-({[4-(5-trifluoromethylpyridin-2-yl)piperazino]sulfonyl}methyl)-3-(pyrimidin-2-yl)propyl]-N-hydroxyformamide

1^(st) enantiomer eluted M+H 489.5.

2^(nd) enantiomer eluted M+H 489.5.

The racemic starting material was prepared in Example 3.

N-[(1S)-1-({[4-(5-bromopyridin-2-yl)piperazino]sulfonyl}methyl)-4-(pyrimidin-2-yl)butyl]-N-hydroxyformamideandN-[(1R)-1-({[4-(5-bromopyridin-2-yl)piperazino]sulfonyl}methyl)-4-(pyrimidin-2-yl)butyl]-N-hydroxyformamide

1^(st) enantiomer eluted M+H 513/515.

2^(nd) enantiomer eluted M+H 513/515.

The racemic starting material was prepared using the method outlined inExample 2: M+H 513/515.

EXAMPLE 8

The following compounds were prepared

Prepared using method in B X R1 mpt M + H Example (5-Cl-2-pyridyl)oxy C2-PyrimidinylCH2CH2CH2 (S enantiomer) 484 4 5-CF3-2-Pyridyl N2-PyrimidinylCH2CH2CH2 (S enantiomer) 141–142 503 4 4-F-Phenyl N2-(5-F-Pyrimidinyl)CH2CH2 (S enantiomer) 456.24 6** 4-F-Phenyl N2-(5-F-Pyrimidinyl)CH2CH2 456.2 2 4-Br—Ph N 2-PyrazinylCH(CH3)CH2 mixeddiastereomers 3:1 (A:B) 512 1 4-Cl—Ph C 2-PyrazinylCH(CH3)CH2Diastereomer A 467 1 4-Cl—Ph C 2-PyrazinylCH(CH3)CH2 mixed diastereomers1:2 (A:B) 467 1 4-Br—Ph C 2-PyrazinylCH(CH3)CH2 mixed diastereomers 3:1(A:B) 511 1 5-Cl-2-Pyridyl N 5-F-2-PyrimidinylCH(CH3)CH2 mixeddiastereomers 1:2 (A:B) 487 1 4-Cl—Ph N 5-F-2-PyrimidinylCH(CH3)CH2Diastereomer A 157–9 1 4-Cl—Ph N 5-F-2-PyrimidinylCH(CH3)CH2Diastereomer B 164–7 1 4-Br—Ph N 5-F-2-PyrimidinylCH(CH3)CH2Diastereomer A 167–9 1 4-Br—Ph N 5-F-2-PyrimidinylCH(CH3)CH2Diastereomer B 183–5 1 4-Cl—Ph C 5-F-2-PyrimidinylCH(CH3)CH2Diastereomer A 195–8 1 4-Cl—Ph C 5-F-2-PyrimidinylCH(CH3)CH2Diastereomer B 155–8 1 3,4-di-Cl—Ph N 5-F-2-PyrimidinylCH(CH3)CH2Diastereomer A 172–3 1 3,4-di-Cl—Ph N 5-F-2-PyrimidinylCH(CH3)CH2Diastereomer B 172–3 1 5-CN-2-Pyridyl N 5-F-2-PyrimidinylCH(CH3)CH2Diastereomer A 478 1 4-F—Ph N (S) 5-F-2-PyrimidinylCH(CH3)CH2 (Senantiomer) 470 7 4-F—Ph N (R,S)-PyrazinylCH(CH3)CH2 (S enantiomer) 4524In the above Table:

** indicates the compound (S enantiomer) prepared by the method inExample 6 using column Chiralpak AD (250 mm×4.6 mm) No. ADooCE-JJ122 andeluent MeOH/MeCN 15/85;

Diastereomers A and B refer to the order of elution from a silica columneluted with 3–5% ethanol in dichloromethane (diastereomer A is the firstfraction to elute, diastereomer B the second).

EXAMPLE 9

We provide NMR data for the following compounds listed in Example 8:

N-[(1S-1-({[4-(5-trifluoromethylpyridin-2-yl)piperazino]sulfonyl}methyl)-4-(pyrimidin-2-yl)butyl]-N-hydroxyformamide

NMR CDCl₃ δ 10.1 (br s, 1H)*; 8.7 (m, 2H); 8.5 (s, 1H)*; 8.4 (br s, 1H);8.1 (s, 1H)*; 7.7 (dd, 1H); 7.2 (m, 1H); 6.7 (d, 1H); 4.9 (m, 1H)*; 4.2(m, 1H)*; 3.9–3.7 (m, 4H); 3.6 (m, 1H)*; 3.4–3.2 (m, 4H); 3.3 (m, 1H)*;3.1–2.9 (m, 3H); 2.0–1.6 (m, 4H). * rotameric signals.

N-({[4-fluorophenylpiperazino]sulphonyl}methyl)-3-[(5-fluoropyrimidin-2-yl)propyl]-N-hydroxyformamide

¹H NMR (DMSO, 373K): 9.46 (br s, 1 H), 8.73 (s, 2 H), 7.08–6.96 (m, 4H),4.42 (br s, 1H), 3.50 (dd, J=14.8, 7.5 Hz, 1H), 3.35 (m, 4H), 3.28 (dd,J=14.8, 4.4 Hz, 1H), 3.18 (m, 4H), 2.97 (m, 2H), 2.21 (m, 2H).

N-[(1R or 1S)-({[4-chlorophenylpiperazino]sulphonyl}methyl)-3-[(3R or3S)-(5-fluoropyrimidin-2-yl)butyl]-N-hydroxyformamide (singlediastereomer A)

1H NMR (CDCl3) (2 rotamers in approximately equal proportions): 8.72 (s,0.5H), 8.57 (d, 2H), 8.25 (s, 0.5H), 7.89 (s, 0.5H), 7.23 (dd, 2H), 6.83(dd, 2H), 4.94 (sext, 0.5H), 4.30 (m, 0.5H), 3.57 (dd, 0.5H), 3.44 (m,2H), 3.37 (m, 2.5H), 3.16 (m, 5.5H), 3.02 (dd, 0.5H), 2.52 (ddd, 0.5H),2.35 (ddd, 0.5H), 2.02 (dt, 0.5H), 1.89 (ddd, 0.5H), 1.40 (dd, 3H);

N-[(1R or 1S)-({[4-bromophenylpiperazino]sulphonyl}methyl)-3-[(3R or3S)-(5-fluoropyrimidin-2-yl)butyl]-N-hydroxyformamide (singlediastereomer A)

¹H NMR (CDCl3) (2 rotamers in approximately equal proportions): 8.72 (s,0.5H), 8.57 (d, 2H), 8.25 (s, 0.5H), 7.89 (s, 0.5H), 7.38 (dd, 2H), 6.80(dd, 2H), 4.94 (sext, 0.5H), 4.30 (m, 0.5H), 3.57 (dd, 0.5H), 3.44 (m,2H), 3.37 (m, 2.5H), 3.16 (m, 5.5H), 3.02 (dd, 0.5H), 2.52 (ddd, 0.5H),2.35 (ddd, 0.5H), 2.02 (dt, 0.5H), 1.89 (dt, 0.5H), 1.40 (dd, 3H);

N-[(1R or 1S)-({[4-chlorophenylpiperidino]sulphonyl}methyl)-3-[(3R or3S)-(5-fluoropyrimidin-2-yl)butyl]-N-hydroxyformamide (singlediastereomer A)

1H NMR (CDCl3) (2 rotamers in approximately equal proportions): 8.69 (s,0.5H), 8.57 (d, 2H), 8.25 (s, 0.5H), 7.89 (s, 0.5H), 7.27 (obscured),7.13 (dd, 2H), 4.91 (sext, 0.5H), 4.30 (m, 0.5H), 3.87 (m, 2H), 3.57(dd, 0.5H), 3.35 (dd, 0.5H), 3.18 (m, 1.5H), 3.00 (dd, 0.5H), 2.85 (m,2H), 2.55 (m, 1.5H), 2.35 (ddd, 0.5H), 2.06 (dt, 0.5H), 1.88 (m, 2.5H),1.7 (obscured), 1.40 (dd, 3H);

N-[(1R or 1S)-({[3,4-dichlorophenylpiperazino]sulphonyl}methyl)-3-[(3Ror 3S)-(5-fluoropyrimidin-2-yl)butyl]-N-hydroxyformamide (singlediastereomer A)

1H NMR (CDCl3) (2 rotamers in approximately equal proportions): 8.62 (s,0.5H), 8.55 (d, 2H), 8.22 (s, 0.5H), 7.86 (s, 0.5H), 7.28 (m, 1H), 6.95(m, 1H), 6.73 (m, 1H), 4.92 (sext, 0.5H), 4.30 (m, 0.5H), 3.57 (dd,0.5H), 3.44 (m, 2H), 3.37 (m, 2.5H), 3.16 (m, 5.5H), 3.02 (dd, 0.5H),2.52 (ddd, 0.5H), 2.37 (ddd, 0.5H), 2.04 (dt, 0.5H), 1.89 (dt, 0.5H),1.40 (dd, 3H);

N-[(1R or1S)-({[4-(5-cyanopyridin-2-yl)piperazino]sulphonyl}methyl)-3-[(3R or3S)-(5-fluoropyrimidin-2-yl)butyl]-N-hydroxyformamide (singlediastereomer A)

1H NMR (CDCl3) (2 rotamers in approximately equal proportions): 8.72 (s,0.5H), 8.55 (s, 2H), 8.41 (s, 1H), 8.22 (s, 0.5H), 7.86 (s, 0.5H), 7.65(m, 1H), 6.61 (dd, 1H), 4.92 (m, 0.5H), 4.30 (m, 0.5H), 3.78 (m, 4H),3.57 (dd, 0.5H), 3.38 (m, 2H), 3.30 (m, 2.5H), 3.16 (m, 1.5H), 3.02 (dd,0.5H), 2.52 (m, 0.5H), 2.37 (m, 0.5H), 2.04 (dt, 0.5H), 1.84 (dt, 0.5H),1.40 (dd, 3H);

N-[(1S-({[4-(4-fluorophenylpiperazino]sulphonyl}methyl)-3-[(3S)-(5-fluoropyrimidin-2-yl)butyl]-N-hydroxyformamide

1H NMR (DMSO-d6): 9.9, 9.53 (2s, 1H), 8.78 (s, 2H), 7.98 (d, 1H),7.12–6.91 (m, 4H), 4.8, 4.17 (2s, 1H), 3.13 (m, 4H), 3.0 (m, 1H), 1.86(m, 1H), 1.22 (m, 3H).

EXAMPLE 101-({[4-(4-chlorophenyl)piperazin-1-yl]sulfonyl}methyl)-3-(5-chloropyridin-3-yl)propyl(hydroxy)formamide

To formic acid (400 μl, 10.8 mmol) at 0° C. was added acetic anhydride(102 μl, 1.1 mmol) and the mixture was then stirred at RT for 15minutes. The mixture was then re-cooled to 0° C., and a solution of1-(4-chlorophenyl)-4-{[4-(5-chloropyridin-3-yl)-2-(hydroxyamino)butyl]sulfonyl}piperazine(100 mg, 0.22 mmol) in THF was added dropwise via syringe. Afterstirring at RT for 1.5 hours, volatiles were removed in vacuo, and theresidue was azeotroped with toluene (2 mL). The residue was thendissolved in methanol (5 mL) and stirred at 40° C. for 1 hour. Aftercooling to RT, the solvent was evaporated, and the residue dissolved inmethanol (0.5 mL). Diethyl ether (5 mL) was then added and the cloudysuspension stirred at RT for 1 hour. The solid that precipitated wasfiltered, washed with diethyl ether and dried in vacuo, to give thetitle compound as an off-white solid (48 mg, 0.099 mmol).

¹H NMR (DMSO, 373K): 9.55 (br s, 1 H), 8.43 (d, 1 H); 8.41 (d, 1 H),8.17 (br s, 1H), 7.76 (dd, 1 H), 7.25 (m, 2 H), 6.96 (m, 2H), 4.35 (brs, 1H), 3.49 (dd, 1 H), 3.34 (m, 4 H), 3.25 (m, 5 H), 2.67 (m, 2 H),2.02 (m, 2 H). MS (ESI): 487.06, 489.04, 490.08 (MH⁺ 2×Cl).

The starting material was prepared as follows:

(i) ethyl 3-(5-chloropyridin-3-yl)propanoate

To a stirred solution of ethyl(2E)-3-(5-chloropyridin-3-yl)prop-2-enoate (338 mg, 1.6 mmol) [CASnumber 163083-45-2] in dry ethanol (10 mL) at 0° C. under an atmosphereof argon was added solid sodium borohydride (67 mg, 1.75 mmol). Thereaction mixture was allowed to warm to room temperature and stirred forfour hours, whereupon additional sodium borohydride (67 mg, 1.75 mmol)was added. After stirring for an additional eighteen hours, saturatedaqueous ammonium chloride solution (5 mL) was added. Volatiles wereremoved in vacuo, and the residue partitioned between water (10 mL) andethyl acetate (10 mL). The layers were separated and the aqueous phaseextracted with ethyl acetate (3×10 mL). The combined organic extractswere then dried (MgSO₄), filtered and concentrated in vacuo. Flashchromatography (silica gel, 20% to 100% ethyl acetate in hexane) gavethe title compound (132 mg, 0.62 mmol) and the saturated alcohol (70mg).

¹H NMR (CDCl₃): 8.43 (m, 1 H), 8.34 (m, 1H), 7.55 (m, 1H), 4.16 (q, 2H),2.96 (dd, 2H), 2.63 (dd, 2H).

(ii)1-{[4-(4-chlorophenyl)piperazin-1-yl]sulfonyl}-4-(5-chloropyridin-3-yl)butan-2-one

To a stirred solution of 1-(4-chlorophenyl)-4-(methylsulfonyl)piperazine(235 mg, 0.85 mmol) in dry THF (7.5 mL) at −10° C. under an argonatmosphere was added dropwise over 4 minutes a solution of LiHMDS (1.71mL of a 1.0 M solution in THF, 1.71 mmol). The solution was then stirredat this temperature for 40 minutes. A solution of ethyl3-(5-chloropyridin-3-yl)propanoate (201 mg, 0.94 mmol) in THF (1 mL) wasthen added dropwise via cannula over a period of 5 minutes. The reactionwas stirred at −10° C. for an additional 30 minutes before beingquenched with saturated aqueous ammonium chloride solution (5 mL).Volatiles were removed in vacuo, and the residue was extracted withCH₂Cl₂ (3×5 mL). The combined organic extracts were washed with water(10 mL) and brine (10 mL) before being dried, (MgSO₄), filtered andconcentrated in vacuo. Flash chromatography (silica gel, 50% ethylacetate in hexane) gave the title compound (228 mg, 0.52 mmol) andrecovered ethyl 3-(5-chloropyridin-3-yl)propanoate (74 mg, 0.35 mmol).

¹H NMR (CDCl₃): 8.46 (m, 1 H), 8.38 (m, 1 H), 7.58 (m, 1 H), 7.21 (m, 2H), 6.83 (m, 2 H), 3.96 (s, 2 H), 3.37 (m, 4 H), 3.17 (m, 6 H), 2.95(dd, 2 H), MS (EST): 442.07, 444.06, 445.1 (MH⁺ 2×Cl).

(iii)1-1-{[4-(4-chlorophenyl)piperazin-1-yl]sulfonyl}-4-(5-chloropyridin-3-yl)butan-2-ol

To a stirred solution of1-{[4-(4-chlorophenyl)piperazin-1-yl]sulfonyl}-4-(5-chloropyridin-3-yl)butan-2-one(228 mg, 0.51 mmol) in a mixed solvent system of CH₂Cl₂/MeOH (1:1, 5 mL)at RT was added solid sodium borohydride in one portion. The reactionwas stirred for 40 minutes before being quenched with aqueoushydrochloric acid (1 M, 2 mL). The layers were then separated and theaqueous phase extracted with CH₂Cl₂ (3×5 mL). The combined organicextracts were dried, (MgSO₄), filtered and concentrated in vacuo. Thecrude product was then filtered through a plug of silica gel, elutingwith 50% ethyl acetate in hexane to give the title compound (111 mg,0.25 mmol).

¹H NMR (CDCl₃): 8.47 (m, 1 H), 8.40 (m, 1 H), 7.59 (m, 1 H), 7.21 (m,2H), 6.86 (m, 2 H), 4.21 (m, 1 H), 3.45 (m, 4 H), 3.24 (m, 4 H), 3.11(m, 2H), 2.88 (m, 2 H), 1.89 (m, 2 H).

(iv)1-(4-chlorophenyl)-4-{[(1E)-4-(5-chloropyridin-3-yl)but-1-enyl]sulfonyl}piperazine

To a stirred solution of1-{[4-(4-chlorophenyl)piperazin-1-yl]sulfonyl}-4-(5-chloropyridin-3-yl)butan-2-ol(111 mg, 0.25 mmol) in dry CH₂Cl₂ (2.5 mL) at RT was added under anatmosphere of argon, trimethylamine hydrochloride (2 mg, 0.02 mmol),triethylamine (52 μl, 0.25 mmol), then methanesulfonyl chloride (21 μl,0.25 mmol). The reaction was stirred for 30 mins at RT, then quenched byaddition of saturated aqueous sodium bicarbonate solution (5 mL). Thelayers were separated and the aqueous phase extracted with ethyl acetate(3×6 mL). The combined organic extracts were then dried, (MgSO₄),filtered and concentrated in vacuo. The residue was then dissolved inCH₂Cl₂ (2.5 mL) and treated with triethylamine (100 μl, 1.36 mmol).After 30 minutes, the reaction was quenched by addition of saturatedaqueous sodium bicarbonate solution (5 mL). The layers were separatedand the aqueous phase extracted with ethyl acetate (3×6 mL). Thecombined organic extracts were then dried, (MgSO₄), filtered andconcentrated in vacuo. The crude material was used in the next step.

MS (ES): 446.06, 428.06, 430.07 (MH⁺ 2×Cl)

(v)1-(4-chlorophenyl)-4-{[4-(5-chloropyridin-3-yl)-2-(hydroxyamino)butyl]sulfonyl}piperazine

To a stirred solution of1-(4-chlorophenyl)-4-{[(1E)-4-(5-chloropyridin-3-yl)but-1-enyl]sulfonyl}piperazine(crude from previous step), in THF (10 mL) at RT was added a solution ofhydroxylamine (2 mL, 50% aqueous solution in water). The reaction wasstirred for 3 hours at RT before being quenched with saturated aqueousammonium chloride solution (5 mL). The layers were separated and theaqueous phase extracted with ethyl acetate (3×10 mL). The combinedorganic extracts were then dried, (MgSO₄), filtered and concentrated invacuo. The residue was then purified by flash chromatography (silica,100% ethyl acetate) to give the title compound (100 mg, 0.22 mmol).

1. A compound of the formula I, or a pharmaceutically acceptable salt,or an in vivo hydrolysable ester thereof,

wherein B is a 2-pyridyl or 2-pyridyloxy group monosubstituted at the4-, 5-, or 6-position by trifluoromethyl; X is a carbon atom; R₁ is atrimethyl-1-hydantoin C₂₋₄alkyl or a trimethyl-3-hydantoin C₂₋₄alkylgroup; or R₁ is phenyl or C₂₋₄alkylphenyl monosubstituted at the 3- or4-position by halogen, trifluoromethyl, C₁₋₃alkyl, or C₁₋₃alkoxy; or R₁is phenyl-SO₂NHC₂₋₄alkyl; or R₁ is 2-pyridyl or 2-pyridyl C₂₋₄alkyl; orR₁ is 3-pyridyl or 3-pyridyl C₂₋₄alkyl; or R₁ is 2-pyrimidine-SCH₂CH₂;or R₁ is 2- or 4-pyrimidinyl C₂₋₄alkyl optionally monosubstituted by oneof halogen, trifluoromethyl, C₁₋₃alkyl, C₁₋₃alkyloxy, 2-pyrazinyloptionally substituted by halogen, or 2-pyrazinylC₂₋₄alkyl optionallysubstituted by halogen.
 2. A compound as claimed in claim 1, or apharmaceutically acceptable salt, or an in vivo hydrolysable esterthereof, wherein: B is a 2-pyridyl or 2-pyridyloxy group monosubstitutedat the 5- or 6-position by trifluoromethyl; X is a carbon atom; R₁ is atrimethyl-1-hydantoin C₂₋₄alkyl or a trimethyl-3-hydantoin C₂₋₄alkylgroup; or R₁ is phenyl or C₂₋₄alkylphenyl monosubstituted at the 3- or4-position by halogen, trifluoromethyl, C₁₋₃alkyl, or C₁₋₃alkoxy; or R₁is phenyl-SO₂NHC₂₋₄alkyl; or R₁ is 2-pyridyl or 2-pyridylC₂₋₄alkyl; orR₁ is 3-pyridyl or 3-pyridyl C₂₋₄alkyl; or R₁ is 2-pyrimidine-SCH₂CH₂;or R₁ is 2- or 4-pyrimidinyl C₂₋₄alkyl optionally monosubstituted by oneof halogen, trifluoromethyl, C₁₋₃alkyl, C₁₋₃alkyloxy, or 2-pyrazinyl or2-pyrazinyl C₂₋₄alkyl.
 3. A compound as claimed in claim 1, or apharmaceutically acceptable salt, or an in vivo hydrolysable esterthereof, wherein B is selected from 4-chlorophenyl, 4-fluorophenyl,4-bromophenyl, 4-trifluorophenyl, 5-chloro-2-pyridyl, 5-bromo-2-pyridyl,5-fluoro-2-pyridyl, 5-trifluoromethyl-2-pyridyl, 5-cyano-2-pyridyl, and5-methyl-2-pyridyl.
 4. A compound as claimed in claim 3, or apharmaceutically acceptable salt, or an in vivo hydrolysable esterthereof, wherein B is 4-fluorophenyl, 5-chloro-2-pyridyl, or5-trifluoromethyl-2-pyridyl.
 5. A compound as claimed in any one of theprevious claims, or a pharmaceutically acceptable salt, or an in vivohydrolysable ester thereof, wherein R₁ is selected from 3-chlorophenyl,4-chlorophenyl, 3-pyridyl, 2-pyridylpropyl, 2- or 4-pyrimidinylethyl(optionally monosubstituted by fluorine), 2- or 4-pyrimidinylpropyl, and2-(2-pyrimidinyl)propyl (optionally monosubstitued by fluorine).
 6. Acompound as claimed in claim 5, or a pharmaceutically acceptable salt,or an in vivo hydrolysable ester thereof wherein R₁ is2-pyrimidinylpropyl, 2-(2-pyrimidinyl)propyl (optionally monosubstituedby fluorine), or 5-fluoro-2-pyrimidinylethyl.
 7. A compound as claimedin claim 1, or a pharmaceutically acceptable salt, or an in vivohydrolysable ester thereof, wherein the compound is N-[(1R or1S)-({[4-chlorophenylpiperidino]sulfonyl}methyl)-3-[(3R or3S)-(5-fluoropyrimidin-2-yl)butyl]N-hydroxyformamide.
 8. A compound asclaimed in claim 1, or a pharmaceutically acceptable salt, or an in vivohydrolysable ester thereof, wherein the compound of formula I is themost active enantiomer.
 9. A compound as claimed in claim 1, or apharmaceutically acceptable salt, or an in vivo hydrolysable esterthereof, wherein the compound of formula I is the S enantiomer or theS,S enantiomer.
 10. A pharmaceutical composition which comprises acompound as claimed in claim 1, or a pharmaceutically acceptable salt,or an in vivo hydrolysable ester thereof, and a pharmaceuticallyacceptable carrier.
 11. A method of preparing a medicament comprising asclaimed in claim 1, or a pharmaceutically acceptable salt, or in vivohydrolysable precursor thereof, comprising combining the compound with apharmaceutically acceptable diluent or carrier.
 12. A method of treatingarthritis, comprising administering a therapeutically effective amountof a compound as claimed in claim 1, or a pharmaceutically acceptablesalt, or in vivo hydrolysable precursor thereof, to a subject in need ofsuch treatment.
 13. A method of treating atherosclerosis, comprisingadministering a therapeutically effective amount of a compound asclaimed in claim 1, or a pharmaceutically acceptable salt, or in vivohydrolysable precursor thereof, to a subject in need of such treatment.14. A process for preparing a compound as claimed in claim 1, or apharmaceutically acceptable salt, or in vivo hydrolysable ester thereof,which process comprises reacting a compound of the formula II with acompound of the formula R₁CHO to yield an alkene of the formula III,converting the alkene to a compound of the formula IV, and thenconverting the compound of formula IV to a compound of the formula I,and optionally thereafter forming a pharmaceutically acceptable salt orin vivo hydrolysable ester of the compound of formula I, all as set outbelow:


15. A process for preparing a compound as claimed in claim 1, or apharmaceutically acceptable salt, or in vivo hydrolysable ester thereof,which process comprises reacting a compound of the formula II with acompound of the formula R₁COOR to yield a compound of the formula VIII,converting the compound of formula VIII to a compound of the formula IX,converting the compound of formula IX to an alkene of the formula III,converting the alkene to a compound of the formula IV, and thenconverting the compound of formula IV to a compound of the formula I;and optionally thereafter forming a pharmaceutically acceptable salt orin vivo hydrolysable ester of the compound of formula I, all as set outbelow: